Parasitology

Naegleria fowleri

- "Primary amebic meningoencephalitis" (PAM) brain eating amebae • Most commonly affects healthy children and young adults after contact with freshwater • 7-10 day incubation • Symptoms mimic bacterial meningitis - Severe bifrontal headache, fever, stiff neck, nausea and vomiting, rapid neurologic decline increased neutrophils, increased protein decreased glucose in CSF • Usually fatal within 1 week of onset mortality is 97% can be flagiated without nutrients, infections occur though nose, key can use Miltefosine to treat Laboratory diagnosis: Naegleria - Finding motile amebic trophs in CSF is diagnostic • Cysts are not seen • 10-25 μm • Explosively extends large pseudopods for movement (wet mount) • Nucleus contains a large karyosome halo surrounding it movement is lobopodian, seen at autopsy - Culture • Add a drop of CSF sediment to a plate of non-nutrient agar with E. coli growing in a lawn • Incubate at 37°C • Observe microscopically each day for 7 days, looking for thin tracks in the agar - Enflagellation • Organism can be induced to change into a flagellated troph lack of nutrients causes this • 1 drop of CSF sediment is added to 1 mL distilled water and incubated at 37°C for 2-20 hours - Direct immunofluorescence - Molecular - Cell culture

Plasmodium

- 156 named species of RBC parasites which infect a wide variety of animals - 5 that infect humans • P. falciparum, P. vivax, P. ovale, P. malariae these 4 most common - P. knowlesi is zoonotic (monkeys) but can affect humans • Transmitted by Anopheles mosquitoes • 247 million cases of malaria in 2008 (WHO) with about 1 million deaths, mostly of children in Africa - Chloroquine is the drug of choice for treatment and prophylaxis

Leishmania: Lab diagnosis

- Amastigote is the diagnostic stage • Found in macrophages from the periphery of skin lesions and intracellularly in cells from bone marrow, liver or spleen biopsy replicate inside macrophage • Wright's stain • 2-5 μm with pale blue cytoplasm, a large red nucleus and a rodlike kinetoplast in the cytoplasm look like tiny WBCs inside a WBC - Serologic testing may be useful if patient is from a non-endemic area

Apicomplexa: Plasmodium • Laboratory diagnosis

- Blood smears • Giemsa stain • Thick smears used for detection of parasites • Thin smears are examined for determination of species - Morphology of both infected RBCs and parasites is important for diagnosis to species - Trophozoites, schizonts and gametocytes may be seen must note which stages are present, can see certain shapes that help differentiate, also note travel history

Acanthamoeba spp.

- Chronic granulomatous amebic encephalitis (GAE) • Immunocompromised and chronically ill populations • Thought to spread from lungs to CNS through blood • Insidious onset very gradual • Symptoms include headache, confusion, dizziness, drowsiness, seizures, sometimes hemiparesis - Keratitis • Painful vision-threatening disease of the cornea infection • Often associated with extended wear soft-contacts misdiagnosed as herpes simplex or bacterial infection, leads to death or blindness, Aids patients with CD4 count less than 250/mL cutaneous lesions risk factors include homemade saline solution and frequent infection and swimming with contacts in • Laboratory diagnosis: Acanthamoeba - Cysts and trophs can be seen in brain biopsy and corneal scrapings - Cultured in the same manner as N. fowleri - Cell culture - Serology by complement fixation • Treatment - Fluconazole, SXT-bactrim, pentamidine isethionate antifungal also used for leishmania Trophs are 20-45 μm, have a single nucleus with a large karyosome, may exhibit "spine-like" projections acanthopodia Cyst walls are wrinkled in appearance

T. brucei: Lab Diagnosis

- Definitive diagnosis requires finding the trypomastigotes in blood, lymph node aspirate, bone marrow or CSF • 15-20 μm, single nucleus, flagellum and undulating membrane attach to kinetoplast, extracellular - ELISA tests are available for direct detection and serology - CATT and latex screening tests for antibodies Card agglutination test for trypanosomes - IFA serology - PCR in development

T. brucei rhodesiense

- East African sleeping sickness much less common, more rapid • Chancre may develop at bite site • Fever, headache, muscle & joint aches and enlarged lymph nodes develop in 1-2 weeks post bite • Infects the CNS in several weeks leading to mental deterioration and other neurological problems • Death occurs within months when multiple organs are infected lots of trypomastigotes are present more than west, west has less organisms present

Plasmodium Lifecycle 2

- Erythrocytic phase • Merozoites attach to RBCs and enter the cell through invagination of the red cell membrane • Organisms feed on hemoglobin • Early trophozoites are vacuolated, ring shaped and uninucleate presence or absence can help speciate- Excess protein, an iron porphyrin and hemin combine to form malarial pigment can cause damage to infected human depending on species • Troph continues to mature and becomes a schizont when the nucleus begins to divide • Mature schizonts contain multiple merozoites which are then released into circulation - Either destroyed by the immune system or reinfect RBCs • After several erythrocytic cycles, the merozoites develop into gametocytes instead of schizonts - Macrogametocytes are the female form - Microgametocytes are the male form • Mosquito ingests gametocytes during a blood meal

Giardia duodenalis 2

- Infections • Many are asymptomatic • 12-14 day incubation • Trophs colonize the duodenum by adhering to columnar epithelial cells with ventral sucking disks - Irritates and damages intestinal mucosa causes sever irritation - Interferes with absorbance of nutrients • Acute- self-limiting with malaise, cramps, nausea and diarrhea lasting 1-4 weeks - "explosive", foul-smelling, non-bloody diarrhea • Chronic- episodes of recurrent diarrhea, abdominal distention and discomfort, belching and heartburn - Can lead to dehydration, malabsorption and impairment of pancreatic function up to 20% weight loss, symptoms can last for several months - Treatment • Metronidazole is the current drug of choice • Albendazole may also be used - Diagnosis • Multiple stool specimens • Duodenal aspirate or Enterotest (string test) is used when symptoms persist and organism is not found in stool specimens 3 stool specimens over 10 days • Trophs- 9-21 μm x 5-15 μm, "falling-leaf" motility, tear-drop shape, 6 flagella, 2 nuclei with small central karyosomes • Cysts- 8-12 μm x 7-10 μm, oval to round, 4 nuclei usually seen at one end with small karyosomes, cytoplasm may pull away from the cell wall and contains 4 median bodies see face with giardia slide 37 - Diagnosis • Antigen detection - EIA, DFA, Immunochromatography - Have high sensitivity and specificity - Multiple specimens may still be required • PCR tests are available - Multiplex PCR used for strain typing in epidemiologic studies

Non-pathogenic organisms

- Must be differentiated from E. histolytica • See Table 28.3, page 626 for descriptions of trophs and cysts - Entamoeba coli reference slide 16 pp 2 • Distinguished from E. histolytica by having an eccentric karyosome, and mature cysts with 8 nuclei. If chromatoidal bodies are present, they have splintered ends, rather than rounded as in E. histolytica • Non-pathogenic amebae - Entamoeba hartmanni small race version • Very similar to E. histolytica in structure of both trophs and cysts nondirectional motility • Size differentiates - Endolimax nana slide 18 • A very small amoeba (5-12um) with a large, eccentric karyosome and thin nuclear envelope • Mature cysts contain 4 nuclei - Iodamoeba bűtschlii slide 19 • Both the trophozoite and cyst have one nucleus with a large karyoosome • Cyst contains a large glycogen vacuole, key

Apicomplexa • Plasmodium Disease 4

- P. falciparum most prevalent in sub Saharan Africa • Invades RBCs of all ages • Infection does not become periodic as early in infection and is usually less than 48 hours when established has smallest delicate rings, has chromatin dots across from each other look like head phones. can infect cells multiple times troph can be pushed up against the side of the RBCs • Untreated primary infection usually ends within 2 to 3 weeks • Complications - Cerebral malaria - Renal failure ("Blackwater fever") see intravascular hemolysis, sever anemia present - Acute lung injury - DIC, hypotensive shock parasitemia can exceed 50% of RBCs

Apicomplexa • Plasmodium Disease 3

- P. malariae • Infects older RBCs infects the smaller ones • Longer incubation period (18-40 days) • Regular, 72 hour periodicity from the beginning quartan life cycle • Chronic infection with low-grade parasitemia can last for years related to the coarse malaria pigment that is produce and lead to nephritic syndrome • Antigen/antibody complexes can be deposited in the glomeruli leading to nephrotic syndrome band form is also seen

Apicomplexa • Plasmodium Disease 2

- P. vivax most prevalent species worldwide may see Schuffner dots/stippling , pg 646 • 7-10 day incubation is usual • Only infects reticulocytes, so parasitemia is limited to 2-5% of RBCs infects the larger RBCs • 48 hour cycle of paroxysms ("benign tertian") • Untreated primary attack can last from 3 weeks to 2 months • 50% will relapse in weeks or months- up to 5 years, dormant bodies • Severe complications are rare - P. ovale • Infection is similar to P. vivax very difficult to differentiate between - Less severe, relapses less frequently, spontaneous recovery after 6-10 paroxyms infected RBCs can be oval shaped or slightly fringed pg 648

Plasmodium Disease (Malaria)

- Periodic febrile paroxysm is the classic manifestation of malaria must know cold, hot and sweating stages • Cold stage (sensation of cold, shivering) lasts up to an hour • Hot stage ( fever up to 104°) lasts up to 4 hours, may have vomiting • Sweating stage tiredness and body tries to return to normal temps • Time between paroxysms is usually 48 or 72 hours varies depending on species tertian is 48 hours reinfects RBC on third day, quartian is 72 hours reinfects on 4th day - Other symptoms include myalgias, malaise, headache, splenomegaly, hepatomegaly, anemia, NVD

Blastocystis hominis an opportunist

- Taxonomy and its role in disease are controversial • Has been classified in the past as yeast, fungus and as an amoeba - Currently classified by genetic sequencing into an informal group called Stramenophiles in which the genetic makeup contains aspects of unicellular and multicellular organisms • May cause gastroenteritis - Recurrent diarrhea with no fever, vomiting, abdominal pain - Symptoms may be more pronounced in HIV and other immunocompromised patients

Buffy Coat Smear

- Used to ID L. donovani and trypanosomes - L. donovani intracellular • Will be found in the mononuclear cells in the buffy coat • Nuclear material will stain dark red-purple and the cytoplasm will be light blue - Trypanosomes will congregate around the buffy coat cells extracellular

Leishmania spp. 2

- Visceral (kala-azar)- spreads to the organs of the RE system liver, spleen, lymph nodes and bone marrow • Leishmania donovani complex, L. infantum • Malaise, anorexia, weight loss, headache and fever mimics malaria • Can progress to splenomegaly and hepatomegaly with increased liver enzymes, hypogammaglobulinemia and hypoproteinemia; kidney and heart can be affected • Usually leads to death within 2 years if untreated - Lifecycle refer to handout pg 1 • Promastigote is transmitted to humans through the salivary glands of the sandfly - Phagocytized by macrophages • Converts to amastigote stage and multiplies within the macrophage diagnostic stage • Released when the cell lyses and is ingested by female sandfly - Vector control and bite prevention are important for controlling the spread insecticides and netting

T. brucei gambiense

- West African sleeping sickness • Much slower progression more chronic form • Intermittent fevers, joint & muscle aches, headaches, weight-loss, Winterbottom sign • Evidence of CNS involvement in 1-2 years - Personality changes, daytime sleepiness, progressive confusion, partial paralysis, difficulty walking • Death often occurs about 3 years after the tsetse bite see fewer organisms in the blood

E. histolytica forms

90% are asymptomatic but can still spread it - Dysentery (acute amebic) type A, develops over days to months • General abdominal complaints • Large numbers of diarrheal stools up to 20 per day - Trophs, blood and mucus can be seen in fresh stool see trophs will ingested RBCs most often in this stage also see Charcot crystals brake down of eosinophiles, possible indication of parasitic infections - Colitis (chronic) • Asymptomatic and diarrheal periods will alternate • Amebic granuloma may form in the intestinal submucosa can form one large mass flask shaped ulcers, key histo results - Extraintestinal infections have to survive the hosts immune response but if they do they move to stage C • Organism penetrates through the intestinal mucosa and enters circulation • Abscesses in the right lobe of the liver are most common liver most common - Fever, URQ pain, weight loss, and increased WBCs and liver enzymes - Can be asymptomatic • Other affected sites can include the lungs, can have productive cough, perineal area, bladder, and brain

T. cruzi - Chagas disease: Three stages

Acute - Incubation phase is 2-4 weeks - Can be asymptomatic - Local symptoms including chagoma and edema around the eye (Romaña's sign) can last from 4-8 weeks only if bite was near the eye - Systemic symptoms including fever, lymphadenitis, hepatosplenomegaly, malaise, myalgia, rash, edema of face, legs or feet possible meningoencephalitis in children under 5 or immunocompromised • Latent intermediate phase- Can last from 10-40 years will not detect organisms in the blood, are in different organs • Chronic most common diagnosed - Cardiomyopathy is the most common clinical sign - Megaesophagus or megacolon » Difficulty swallowing, regurgitation, severe constipation - CNS involvement seen in HIV patients » Meningoencephalitis and granulomatous encephalitis can lead to death from multiple organ destruction,

Protozoa: Apicomplexa

Blood and tissue parasites • Complex life cycles - Sexual and asexual phases • Humans are definitive hosts when sexual reproduction occurs in human tissue and are intermediate hosts when asexual reproduction occurs - May require insect vector or intermediate host to complete life-cycles like malaria

Protozoa: Intestinal Amebae

Challenging to identify because differences are subtle - Number & structure of nuclei, other internal structures, size, motility of trophozoites - Can be difficult to distinguish from background material permanent stains are very useful to differentiate • Two pathogens - Entamoeba histolytica and Blastocystis hominis opportunist • Metronidazole is given to treat E. histolytica only interferes with DNA, refer to handout from pp1 Life Cycle - Direct transmission through contaminated food or water - No intermediate hosts - Cysts are the infective stage hard study resistant shell - Trophs colonize the cecum cause the most damage pg 624 reference slides 4 and 5 pp 2 and handouts

Protozoa: Intestinal Amebae • Laboratory diagnosis

Laboratory diagnosis - Stool is the primary specimen • Fresh - Motile trophs may be observed • Preserved - Trichrome stain - Sigmoid biopsy and abscess specimens may also be submitted for externa infection - Entamoeba histolytica • EIA kits are available for direct detection of antigen in stool • Serology (IgG) can be useful for diagnosis of extraintestinal infection

Dientamoeba fragilis

Protozoa: Flagellates • Dientamoeba fragilis - Worldwide distribution - Causes a mild, non-invasive diarrheal illness GI symptoms can or can not cuase symptoms • With fatigue, abdominal pain, nausea May be transmitted in co-infection by Enterobius or Ascaris primary Enterobius - Diagnosed by direct microscopy • No cyst stage has been identified • Trophs- 5-12 μm, amoebae shaped, one to two nuclei, amebae like, karyosome clusters are key, karyosome clusters of 4-8 granules motile by pseudopods, flagella cannot be seen trophs have very short life span

Protozoa: Flagellates • Non-pathogenic

Protozoa: Flagellates • Non-pathogenic intestinal flagellates - Must be differentiated from the pathogens - Chilomastix mesnili • Found worldwide • Trophs- 10-20 μm x 3-10 μm, rotary type motility, one nucleus, spiral groove • Cysts- 6-10 μm, lemon shaped with a protrusion at one end, one nucleus - Pentatrichomonas hominis also called trichomonas hominis • Trophs- 6-14 μm, jerky motion, one nucleus, undulating membrane running entire length, axostyle, jerky motion is key • No cyst stage reference slide 4 pp 3 parasites

Thin Blood Smears

Thin Blood Smears - Smear is prepared in the same manner as for hematology - Fixed in methanol and air dried before staining - Smears are stained with Giemsa (preferred) or Wright stains prior to observation - Shows detailed characteristics of organism morphology - Size of infected RBCs and uninfected RBCs can be compared so we want the RBCs present - % parasitemia should also be calculated and reported (# of infected RBCs)/ (total RBCs counted ), times 100 - Examine entire slide on low power to look for microfilariae - Examine at least 300 fields under oil immersion more can be better - Organisms can be ID'ed to the species level refer to slide 35 pp 1

Apicomplexa: Cystoisospora belli

also called isospora Found primarily in tropical, subtropical and warm temperate regions • Transmitted through contaminated water or food • Infection - Many patients are asymptomatic - Acute infection clinical picture is the same as for C. parvum - Self-limiting and resolves in several weeks - More serious in immunocompromised sever diarrhea and loss lots of weight

Giardia duodenalis

pg 635, look over could also be called G lamblia or G intestinalis - World-wide distribution, affects humans and other animals - Transmitted through contaminated food or water and the fecal-oral route direct transmission • Low infective dose is found in US, risks to people that travel to other places, also called bever fever or travelers/backpacker diarrhea - Cysts survive several months of cold temperatures and resist killing by chlorine and iodine, most common intestinal protozoa in US reference slide 33 cyst contaminates food and water , pass into intestines, stomach acid causes exestuation to occur makes two trophs feed and grow, these both divide, infection is caused, can attach by suction disks to lining of small intestine, causes bleeding and malabsorption, encystation when moving to large intestine occurs and are then passed, cysts can survive in cold

Parasitology

refer to physical handouts

Leishmania spp.

• - Cutaneous leishmaniasis most mild - Firm, painless papule that may be very itchy at or near site of insect bite forms 2 weeks to 2 months after bite • Lesion becomes much larger (2-8 cm) and may ulcerate • Self-limiting but if not treated can lead to other forms of infection • Granulomas sometimes form Bagdad boils • L. tropica, L. major, L. Mexicana complex, etc. L tropica is most common for granulomas - Mucocutaneous infection- untreated primary lesions spread and can destroy the mucosal surfaces of the nose and mouth can occur years after papules had formed, chronic stuffiness or nosebleeds(L. braziliensis)

T. cruzi: Lab diagnosis

• - Demonstration of trypomastigotes in blood • 15-20 μm, single nucleus, anterior flagellum, undulating membrane, often in C or U shapes rounded • Appear about 10 days after infection and persist through the acute phase - Demonstration of amastigotes in tissue - PCR - Serology, serology is not the best • CF, chemiluminescence, IFA, RIA, and ELISA

Trichomonas vaginalis

• - Infects the urogenital tract of both females and males - STD • 7.4 million new cases per year in US (estimate) not reportable in US • Often associated with other STDs and increases the risk of HIV infection inflammatory response causes decrease resistance to other STDs • Males are usually asymptomatic but may develop non gonococcal urethritis with purulent discharge • Females often develop vaginitis common to see females being reinfected by male partners - Treated with metronidazole and/or clindamycin risk factor, multiple sexual partners, poor personal hygiene, and poor people

African Sleeping Sickness

• African Sleeping Sickness - T. brucei gambiense and T. brucei rhodesiense G is 99% of cases west chronic and more mild, R is east is more rapid • Closely related and indistinguishable morphologically - Local inflammatory reaction (chancre) at bite site which resolves in 1-2 weeks can last up to 4 weeks - Two stages • Organism found in peripheral circulation & lymphatics, hemolymphatic • Organism crosses blood-brain barrier hemolymphatic phase see trypomastigotes occur at 1-3 weeks with winterbottom sign swelling of lymph notes , second phase has CNS symptoms after crossing blood brain barrier.

Plasmodium Lifecycle - Mosquito phase

• After ingestion, the microgametocytes develop six to eight flagella (exflagellation) and penetrate the macrogametocytes forming a zygote • Zygotes enter the stomach wall as oökinetes and develop into oöcysts which produce sporozoites • Sporozoites are released and migrate to the mosquitoes' salivary glands, completing the cycle

Balamuthia mandrillaris

• Balamuthia mandrillaris - Cutaneous infections found only in soil but can be inhaled from particles or by direct inoculation • Painless skin nodules and lesions - Encephalitis • Opportunistic GAE with similar progression and symptoms as Acanthamoeba GAE very deadly and fatal - Laboratory diagnosis • Usually diagnosed by finding trophs and cysts in skin lesions or tissue at autopsy • IFA serology methods are available using amoeba-coated slides • Cell culture

Balantidium coli

• Balantidium coli - The only pathogenic ciliate - Transmitted through contaminated food and water - Life cycle is similar to amebae ingested and then passed in stool - Usually asymptomatic, but can cause dysentery and colitis similar to infection by E. histolytica • Can invade the intestinal mucosa, but dissemination is very rare pigs are natural host, humans are accidental host - Laboratory diagnosis • Direct, microscopic examination of stool or biopsy of intestinal mucosa • Troph is oval, 45-60 μm x 30-40 μm OR 90-120 μm x 60-80 μm, ciliated, with a small opening at its posterior, cytopyge • Cyst is spherical or oval, 45-75 μm, cilia are difficult to see within the thick cyst wall • Both with one large kidney-bean shaped macronucleus and one small, round micronucleus (not seen), and vacuolated cytoplasm, reference slide 31 pp 2

Specimens for Blood and Tissue Parasites 2

• Biopsy specimens tissue specimens - Usually needed to diagnose Leishmania - Skin, liver, spleen and bone marrow tissue samples • CSF - Organisms causing amoebic meningitis and sleeping sickness may occasionally be observed in CSF - For detection of Naegleria fowleri, a non-nutrient agar can be seeded with E. coli, overlaid with CSF sediment, sealed, incubated and observed daily for thin tracks in the bacterial growth incubate for 7 days, normally feed on GN bacteria

• Blastocystis hominis

• Blastocystis hominis - Amoebic form • Rarely seen and difficult to identify - Vacuolated form central body form • 5-15 μm, usually round • Characterized by a large central vacuole surrounded by small, multiple nuclei fills most of the internal structure

Specimens for Blood and Tissue Parasites

• Blood smears make 3 thin and 3 thick- Whole blood direct from fingerstick or EDTA • Slides must be made within 1 hour of collection in EDTA otherwise RBC morph will change, purple tube - Giemsa or Wright stains - Used to detect malaria, Babesia, Trypanosoma and some microfilaria • Tissue biopsies - Trichinella, Leishmania and Toxoplasma gondii

Apicomplexa: Toxoplasma gondii 5

• Clinical infection - Presentation depends on immune status of the individual • Immunocompetent persons are often asymptomatic or may have mild, non-specific symptoms mimics flue or mono - After resolution, the organism enters an inactive stage and tissue cysts develop restarts when immune system becomes compromised • Congenital infections occur when organisms cross the placenta and enter fetal circulation - Symptoms vary depending on trimester of infection and may not be evident for months or years - Retinochoroiditis, cerebral calcification, hydrocephalus or microcephaly first trimester leads to miscarriage, third is most likely to cause infection and leads to eye problems but later on in childhood

Apicomplexa: Toxoplasma gondii 6

• Clinical infection - In Immunocompromised patients • Toxoplasmosis in immunocompromised individuals can be due to either a newly acquired infection or reactivation of a latent infection - Encephalitis is the most common manifestation, myocarditis or pneumonitis also occur usually rapidly fatal • Treatment - Not usually treated in immunocompetent patients - Antimicrobials which block the folic acid pathway (SXT) are often used - Clindamycin and a variety of other antimicrobials have also been used not effective against cysts and bradyzoites acutely infected pregnant patients, immunocompromised, HIV, children with congenital issues

Microscopic Examination part 2

• Concentration Techniques - Purpose is to concentrate the parasites and remove as much debris as possible - Increases recovery • Protozoan cysts, oocysts and spores protozoan trophs cant survive this • Helminth eggs and larvae - Specimen can be fresh or preserved in formalin, SAF or MIF - Sedimentation or floatation methods centrifuge to concentrate, cysts larva and eggs formalin ethyl acetate, removes junk from suspension via centrifugation • Sedimentation Method - Concentrates organisms in sediment at the bottom of a centrifuge tube • Useful for observing protozoan cysts, larvae and eggs - Formalin-ethyl acetate sedimentation method (FES) is commonly used • Ethyl acetate extracts debris and fat from stool • Concentration by centrifugation - Wet mounts are then observed FES Method 1. Make a suspension of stool (walnut sized) in 10 mL saline • Filtered, centrifuged, washed with saline until supernatant is clear 2. Add 10% formalin to fix 3. Add 1-2 mL ethyl acetate and vortex 4. Centrifuge • There will be 4 layers, ring around the debris to disconnect and remove everything except sediment

Modified Trichrome Stain 2

• Cytoplasm of cysts and trophozoites is bluegreen and tinged with purple • Nuclear chromatin, chromatoid bodies and RBCs appear red or red-purple • Eggs and larvae stain red • Background material and yeasts stain green

Other Specimens for Intestinal Parasites

• Duodenal aspirates/Entero-Test (string test) - Uses • When clinical symptoms are present but stool examinations are negative • Giardia • Strongyloides larvae • Eggs of Fasciola hepatica, Clonorchis sinensis • Oocysts of Cryptosporidium or Isospora - Entero-Test Method • Gelatin capsule containing a weighted string is swallowed - One end of the string is taped to the side of the patient's mouth • After 4 hours, the string is removed and the mucus is observed microscopically • Sigmoidoscopy specimens - Wet mount for motile trophs and permanent smear • Sputum not common specimen certain organisms standout - Direct wet mounts can be used to see the filariform larvae in a Strongyloides stercoralis hyperinfection and the eggs of Paragonimus westermani lung fluke, sputum is specimen of choice - Permanent smear can be used to ID Entamoeba histolytica in a pulmonary abscess can lead to extraintestinal abscesses

E. histolytica

• E. histolytica - Once thought to have an infective strain and a non-infective strain • Morphologically identical - Intracellular RBCs indicate E. histolytica • Non-infective now classed as E. dispar can still cause mild infection • Are differentiated by surface antigens and DNA diagnostic for E histolytica, ingested RBCs are definitive, without RBCs could be either - Virulence factors • Adherence to and invasion of intestinal mucosa via surface lectins • Causes programmed cell death followed by lysis and phagocytosis of the host cells uses amoebapores protein causes rapid influx of calcium into intestinal cells causing cell death, and proteases that allow for deeper invasion of intestinal wall • Resists effects of complement • Attracts and then kills neutrophils using chemo attractants

• E. histolytica

• E. histolytica - Trophs • 10-50 μm; usually 15-25 μm, invasive strains often >20 μm • Progressive, directional, rapid motility key, very different from non pathogenic • Single nucleus with finely granular, uniform chromatin and a single, compact, central karyosome nucleus looks like bullseye • May contain RBCs diagnostic - Cysts • 10-20 μm, round pg 626 • Four nuclei finely granular, uniform chromatin and a single, compact, central karyosome • Rounded, elongated chromatoidal bars are usually seen

Cellophane Tape Preparation

• For detection of pinworm (Enterobius vermicularis) • Female worm migrates from the anus at night and lays eggs in the perianal area eggs need oxygen to survive, leads to sever itchiness • Perianal area is swabbed with the sticky side of cellophane tape or a collection paddle first thing in the morning before the patient uses the bathroom or bathes • The sticky side of the tape is rolled or placed onto a glass slide and is observed at low and high power for pinworm eggs

Protozoa: Tissue Amebae

• Free-living, soil or water organisms which feed on bacteria in the environment warm stagnant water, soil present, lakes, ponds - Only cause disease when they gain access to human CNS mostly rapidly fatal • Three primary genera - Naegleria, Acanthamoeba, Balamuthia - Others may cause rare cases of CNS disease • Infections are very rare pg 631

Helminths: Trematodes

• Generally flat, fleshy worms except for blood flukes • Two muscular suckers - An oral type is the beginning of an incomplete digestive system - A ventral sucker serves as a means of attachment • All require a mollusk as an intermediate host, snail- key - An asexual reproductive cycle occurs most larva/eggs need water to mature diagnosis is normally through finding eggs, like wet mounts, concentration techniques, dont use zinc sulfates which pops off the lid except for blood flukes who dont have the lids, are hermaphrodites except for blood flukes pg 657-8 for life cycle and comparison, blood flukes are always the exception

Apicomplexa: Cyclospora cayetanensis

• Humans are the only known host • Transmitted by contaminated food and water • Endemic in many parts of the world • Infection - Incubation period of about 1 week - Frequent, watery, non-bloody stools

Modified Acid-Fast Stain

• Identification of acid-fast parasites - Primarily used to aid in recovery and ID of intestinal coccidian oocysts • Specimen can be any fresh or preserved stool • Stains used: - Kinyoun - Ziehl-Neelsen - 1%-3% sulfuric acid is recommended for decolorizing Oocysts appear as magenta stained against a blue grey background • Specimens preserved in formalin may not take up stain as well

Apicomplexa: Babesia microti

• In US, infections are typically found in the Northeast - NY state, CT, Nantucket & Martha's Vineyard Islands - Newly described, but unnamed, related organisms have caused similar disease on the West Coast and Missouri • WA1, CA1 and MO1 strains

Apicomplexa: Cyclospora cayetanensis 2

• Infection - Other symptoms include anorexia, weight loss, abdominal cramping, vomiting, nausea, low-grade fever - In immunocompetent, symptoms resolve in several weeks • May develop a pattern of relapses lasting several months - Immunocompromised patients may have symptoms lasting up to 4 months • SXT is drug of choice for treatment

Microsporidia 2

• Infection - Immunocompetent • Diarrhea, cramps, lack of appetite and fatigue - Immunocompromised • Persistent and debilitating diarrhea • Dissemination to other sites renal failure from UTIs, respiratory infections, encephalitis, really anwhere • Treatment - No completely effective treatment - Albendazole is the drug of choice - Infection often recurs after medication is stopped

Apicomplexa: Cryptosporidium 3

• Infection - Immunocompromised • AIDS, primary immunodeficiency, cancer, transplant Chronic, life-threatening diarrhea up to 50 stools per day, lots of fluid loss, 17L of fluid loss in a day, can last for months to years in some patients • Treatment - Azithromycin, SXT - Relapses often occur after treatment ends - In HIV patients, keeping the CD4 count elevated by HAART therapy is most useful

Apicomplexa: Cryptosporidium 2

• Infection - In developed countries most infected can be asymptomatic • More common in older children and institutionalized elderly • Typically a self-limiting diarrheal disease - Cholera-like, watery diarrhea with fever, headache, nausea, abdominal cramping • Duration of 9-21 days leads to dehydration - In developing countries • Most often affects children <5 yrs • Diarrhea can be of high volume - Fever, abdominal pain, headache, etc • Can reoccur • Growth and development are often affected

Apicomplexa: Babesia microti 3

• Infection - Seasonal- May through September when ticks are active see more infections - Most cases are asymptomatic likely under reported - 1-6 week incubation period - Malaria-like symptoms with no periodicity hot cold and sweaty stages - Complications include anemia, respiratory, liver or kidney failure and DIC with chronic infections • Used to treat with quinine sulfate and clindamycin but now treatment includes azithromycin and atovaquone.

Apicomplexa: Cryptosporidium

• Infects epithelial cells of the stomach, intestine and biliary ducts • 8 species have been detected in humans - C. parvum and C. hominis have been associated with clinical infection • Transmitted through contaminated water or food or by the fecal-oral route - Low infective dose as few as 10 organisms - Outbreaks in daycare centers, through municipal water systems, contaminated well water food and water born, most caused parasite water born infections plus giardia, resist disinfection with chlorine and ammonia

Apicomplexa: Toxoplasma gondii

• Infects most species of warm-blooded animals - Cats (genus Felidae) are the only known definitive host • Serology based epidemiologic studies indicate it is one of the most common infections worldwide • Humans acquire infection in one of several ways - Ingestion of cysts in undercooked meat - Ingestion of oocysts from fecal contamination of hands, food or water have cats - Organ transplant or blood transfusion - Transplacental transmission

Macroscopic Examination

• Intact worms or proglottids (segments) may be observed on the surface formed must be seen 2-4 hours, stored in fridge 24 hours solid doesnt tend to have as many trophs but they live longer, tends to have more cysts, effects of trophs are more seen in soft but dont live as long • Consistency is noted - Liquid, soft, formed • Presence of blood is noted - Black stools indicate bleeding in the upper GI tract - Bright red blood indicates bleeding in the lower portion any blood should have preservation for permanent smear

Apicomplexa: Cystoisospora belli 3

• Laboratory diagnosis - Only detected through microscopy • Concentration methods are useful • Modified acid fast stain • Oocysts are 32 μm x 14 μm - Organisms autofluoresce at 365 nm and 405 nm 2 sporocysts, the cell wall doesnt stain but they do

Apicomplexa: Cryptosporidium 5

• Laboratory diagnosis - Oocysts are very small and difficult to detect • 4-6 μm, may resemble yeast - Modified acid fast positive (red) - EIA - DFA using a labeled, monoclonal antibody against Cryptosporidium specific antigens is often used • Often in combination with a DFA or EIA for Giardia - PCR

Apicomplexa: Toxoplasma gondii 7

• Laboratory diagnosis - Serology tests (IFA and EIA) are the best method • IgM and acute/convalescent titers are necessary to diagnose acute infection - Infections are rarely diagnosed by microscopy • Body fluids and tissues are potential specimens, but are rarely encountered - Tachyzoites are crescent shaped and may be seen free or intracellularly - Antigen detection • FITC labeled antisera has been used to detect tachyzoites in tissue sections - PCR

Microsporidia 4

• Laboratory diagnosis - Specimens include stool, tissue biopsy, urine and CSF, wherever it can go - Electron microscopy is considered the gold-standard - Giemsa stain • Spores are 1-2 μm - Calcofluor white - PCR looks like bacteria

Apicomplexa: Plasmodium 6

• Laboratory diagnosis - Acridine orange fluorescent staining has been used • Mature RBCs have no nuclear material, so the Plasmodia nuclei are easily visualized • Quantitative Buffy Coat system (QBC) • Utilizes acridine orange use microhematocrit tube with the acridine orange, organisms will fluoresce, is a screening test, if detected move to thin smear refer to slide 12

Apicomplexa: Plasmodium 7

• Laboratory diagnosis - Immunochromatography • Rapid dipstick antigen detection - PCR - Serology • IFA tests for each of the four species have been developed • Cross reaction between species is an issue

Apicomplexa: Plasmodium 2

• Laboratory diagnosis: Blood smears - First, rule out P. falciparum sub Saharan Africa • Infections may be heavy, involving 20% or more of the RBCs any ages RBCs, can exceed 50% • Tiny ring forms that occupy less than 1/3 the diameter of the RBCs smallest and most delicate, have 2 dots with headphone like look,most likely to multiply infect a single cell - Often plastered on the RBC cell membrane, known as an "applique" effect • Developing ring forms or schizonts are rarely observed • Presence of banana (crescent) shaped gametocytes is diagnostic (appear after 7-10 days after onset) refer to handout

Apicomplexa: Plasmodium 4

• Laboratory diagnosis: Blood smears - P. malariae affects only mature RBCs • Developing trophs that extend to the borders of the RBC membrane producing "bands" • RBCs are of normal size and are not pale • No Schüffner's dots • Schizonts with no more than 6-12 merozoites arranged in "rosette" form • Malarial pigment is abundant and coarse, refer to slide 8 pp 4

Apicomplexa: Plasmodium 3

• Laboratory diagnosis: Blood smears - If not P. falciparum, consider P. vivax affects retics most often • Irregular, enlarged, pale RBCs with prominent, pink staining granules (Schüffner's dots) • Ring form in all stages of development may be seen • As trophs mature, they begin to fill the RBC with "flowing" or "ameboid" cytoplasm • Schizonts comprised of 12-14 or more merozoites should count them, have the most of all the species • Gametocytes are large and circular • Malarial pigment, seen as finely granular brownish pigment may be abundant in both the gametocyte and the gameteocyte refer to slide 6 pp 4 parasites

Apicomplexa: Plasmodium 5

• Laboratory diagnosis: Blood smears - P. ovale least common, resembles vivax • RBCs are enlarged and may be oval and have fringed edges key, one fringed edge, looks ghost shaped • Schüffner's dots in all stages • Compact pigment • Schizonts contain 8-12 merozoites, refer to slide 10 pp 4

Apicomplexa: Toxoplasma gondii 3

• Life cycle - Humans can become infected in several ways • Consuming food or water contaminated with cat feces or by contaminated environmental samples (such as fecal-contaminated soil or changing the litter box of a pet cat) • Eating undercooked meat of animals harboring tissue cysts • Blood transfusion or organ transplantation • Transplacentally from mother to fetus

Apicomplexa: Toxoplasma gondii 2

• Life cycle - In cats: pg 651 • Both asexual and sexual reproduction takes place in the epithelial cells of the small intestine • Oocysts are formed by sexual reproduction and are released in the feces not initially infective - Require 2-5 days in the environment to become infective - Can survive for more than a year and are resistant to freezing and drying

Apicomplexa: Cyclospora cayetanensis 3

• Life cycle - Oocyst is the infective stage • Require 1 to 2 weeks outside the body to become infective - Excysts in the small intestine, releasing sporozoites which invade the intestinal epithelium - Both asexual (type I) and sexual (type II) forms of merontes develop type 1 are going to produce merozoites that infect other intestinal cells, type 2 proceeds to sexual stage fertilization occurs and zygote forms and oocysts forms and is passed in stool and is not infective until 1-2 weeks • Identification - Oocysts resemble C. parvum but are larger 8-10 um, vs 4-6 um of C parvum - Two sporozoites can also be seen after several days - Modified acid fast stain - Organism autofluoresces

Apicomplexa: Toxoplasma gondii 4

• Life cycle - Sporozoites are released in the intestine and penetrate the intestinal wall, gaining access (now as tachyzoites) to circulation and migrating to various organs - Tachyzoites invade host cells, multiply and cause the cells to rupture, releasing more tachyzoites • Eventually resulting in tissue destruction - Immune response by the T-cells results in the formation of a cyst inside the affected tissue -inside the cyst Tachyzoites convert to bradyzoites which very slowly grow and reproduce during the latent phase infection can remain latent until immune system is compromised if cysts are ingested then the cyst can break open and the bradyzoites turn into tachyzoites which cause infection

Apicomplexa: Babesia microti 4

• Life cycle - Very similar to life cycle of Plasmodium • No exoerythrocytic phase not as complex • Laboratory diagnosis - Wright or Giemsa stained thick and thin blood smears - Only trophozoites will be seen • Multiple organisms may be present in an RBC • Early trophs are small, delicate ring forms with dense chromatin only see rings with babesia, and rings can be seen extracellularly tetras - Can be misidentified as P. falciparum • Mature trophs have a characteristic Maltese cross form - IFA serology tests and PCR have been developed dont see other stages like falciparum or pigment, refer to slide 18 pp 4

Apicomplexa: Cystoisospora belli 2

• Life cycle is similar to Cryptosporidium spp. - Oocysts are not infective until 24-48 hours after being passed in stool can have cyst stage - No autoinfective stage - May have a hypnozoite/cyst stage no thin walled occur, have a thick walled

Apicomplexa: Cryptosporidium 4

• Life cycle pg 653 - Sexual and asexual phases occur in the same individual - Oocysts are infective - Sporozoites are released which enter the brush boarder epithelium of the intestine - Develop into trophozoites and then meronts which produce merozoites which invade other cells when released - Merozoites can also develop into gametes - Fertilization leads to the formation of oocysts which are either released or can autoinfect autoinfection is possible in cryptosporidium note thin and thick wall oocytes can occur, thin will cuase autoinfection by rupture in intestine, thick wall oocysts are fully sporulated, is infective and goes into environment can pass though intestine and infect others patient can remain infective after the fact and continue to shed after diarrhea ceases

Microsporidia 3

• Life cycle starts in GI but can disseminate - Ingested spores pass into the duodenum - Sporoplasm is transferred to the host cell through a polar tube looks like a tail, sporoplasm is internal contents of cell - Once inside the cell, the microsporidia multiply either in a vacuole or freely in the cytoplasm through binary fission (meronts) - Meronts develop into sporonts which form polar tubes and a thick membrane as they mature into spores - Host cell ruptures and spores are released, reference slide 41 pp 4

Microscopic Examination

• Microscope should have an ocular with a micrometer - 0.1 and 0.01 mm divisions - Size of the ova/parasites can be an important characteristic for identification • Direct Wet Mount - Primarily for the detection of motile protozoan trophozoites in liquid stool or sigmoidoscopy specimen - Method • Place one drop of saline and one drop of iodine at opposite ends of a slide • Add one drop of stool to each - May use formalin preserved stool, but not PVA preserved stool PVA turns cloudy when exposed to air - Thin enough to be able to read newsprint through it • Coverslip and seal with nail polish or petroleum jelly - The preparations should not overflow beyond the edges of the coverslips look for motion, trophs are mainly what are being looked for Direct Wet Mount - Method • Examine the entire cover-slipped area first with low power followed by high dry - Observe saline end for helminth eggs and motile trophs - Iodine emphasizes cellular details » Kills trophs

Modified Trichrome Stain

• Modification of the Gomori (Wheatley's) stain • Specimens dont let smear dry before fixing - Smears made from fresh stool must be placed in Schaudinn fixative immediately put on paper towel to remove liquid and then take solid and fix - Specimens from PVA preservative do not need to be fixed - Specimens preserved in SAF do not stain well (should use iron hematoxylin stain instead)

Trichomonas vaginalis - Diagnosis

• No cyst stage look for trophs • Trophs commonly seen in vaginal/urethral wet preps and in urine sediment can die fast and look like WBCs - Mix specimen with one drop of saline and observe under low power (10x) within one hour - Jerky motility, motion of flagella and undulating membrane can be seen only runs half the length - Giemsa stain » Pear-shaped with an axostyle and granules extending the length of the organism » Single nucleus • Culture - Commercial systems including media in a plastic pouch that are examined microscopically for motile trophs » Diamond's media must inoculate within 30 minutes to maintain viablility, hold for 5 days and check every day for organisims swabs can show trichomonas, yeasts, clue cells, WBCs • Antigen detection - Latex agglutination (not yet available in the US) - Direct immunofluorescence - Immunochromatography • DNA probes and PCR

Direct Detection of Antigens

• Nucleic acid assays are rapidly being adopted by clinical laboratories • Enzyme immunoassay - Commercial kits are available - G. lamblia, Cryptosporidium, and Entamoeba are the most common (stool screen) - Malaria EIA test kits are growing in popularity • Direct fluorescent antibody - Monoclonal antibodies are available for Giardia and Cryptosporidium • Serology testing is not widely used, but is available for some organisms - Toxoplasma, Babesia, Trypanosoma cruzi hard to tell between current and past infection in endemic areas. however can be useful for non endemic areas or specific things

Microsporidia

• Obligate intracellular parasites does affect GI system • Primitive eukaryotic organisms which have recently been reassigned to kingdom Fungi more than 1400 species of these 14 infect humans • Worldwide distribution - Host animals include vertebrates and invertebrates • Transmitted through the ingestion of spores that have been shed in the urine and feces of infected animals or other humans

Stool Specimens: "Ova & Parasites"

• Organisms may be shed into stool irregularly, so multiple specimens are often indicated - 3 specimens in a 10 day period is typical single specimen likely wont have it if low numbers are present pooling all specimens is less likely to recover • Clean, dry container should be used - Water and/or urine from toilets and bedpans can destroy trophozoites or introduce free-living protozoa water can have free living parasites, urine can kill • Collection before barium enemas or antimicrobial therapy is started barium treatment causes wait time of 7-10 days, antimicrobials reduce numbers • Delivered to the lab as quickly as possible after collection or placed in preservative • Trophozoites or eggs may disintegrate in a short time must keep organisms viable • Freezing is not acceptable • Preservatives used for stool specimens - PVA (polyvinyl alcohol): also includes mercuric chloride • Permanent smear and DNA -PCR is a two vial system mercuric chloride used for fixation in first vial has PVA resin used to increase adhesion to side, has long shelf life, second is formalin note infective(cysts) stage may not be recover as easily - 10% formalin: may not recover trophozoites • Concentration, direct wet mount and immunoassays no permanent smear - SAF (sodium acetate-acetic acid-formalin]: • Permanent smear and concentration - MIF (merthiolate-iodine-formalin): • Concentration and direct mount

Artifacts

• Other materials seen in stool can be confused with parasites, such as: - Yeasts - Plant cells - Pollen grains - Fungal spores - Plant fibers - Animal hairs - Charcot-Leyden crystals • Found when disintegrating eosinophils are present breakdown product • May or may not represent parasitic infection could be allergies

Introduction

• Parasites are an important cause of morbidity and mortality in human populations world-wide • In the US, we don't always consider them as major causes of disease, but increasingly, they must be considered because: - Rising numbers of immunocompromised patients - Increased travel to countries with endemic parasites - Immigration from endemic areas climate change, population density can increase risks, warmer climates tend to have more, sanitation and public health practices • Symptoms are often non-specific - Vague abdominal discomfort, diarrhea, fever • When physician suspects a parasitic infection, information should be shared with the laboratory to facilitate identification - Patient's particular symptoms - Pertinent information about the patient's recreation, lifestyle and travel, potential eating habits, job • Parasite Groups pg 623 table 28.2 - Protozoa • Amebae, Flagellates, Ciliates, Apicomplexa, Coccidia, Microsporidia - Trophozoites and cysts these are the stages that we encounter, cysts are infective, hardy and survive in environment, trophozoites are feeding forms in host non infective cause damage to host - Helminths • Trematodes flukes • Nematodes round worms - Intestinal, Filaria spread by blood feeding insects • Cestodes • Parasites may rely solely on humans, have an animal reservoir host or be free living during parts of their life cycle refer to handout on body sites

T. cruzi - Life cycle

• Reduvid bug pg 642 - Ingests trypomastigotes during a blood meal will further develop - Epimastigotes develop in the gut then migrate to the salivary glands where they mature into metacyclic trypomastigotes this is infective state • Parasite is transmitted when the reduvid bug defecates in the area of the bite (scratched in) • Humans - Trypomastigotes enter tissue cells where they transform into amastigotes which replicate - Host cells burst, releasing amastigotes » Some reinfect other host cells » Some transform into trypomastigotes this is where tissue damage occurs

T. brucei gambiense and rhodesiense - Life cycle

• T. brucei gambiense and rhodesiense - Life cycle • Tsetse fly ingests trypomastigotes during a blood meal - Long, slender trypomastigote form with a long flagellum • Promastigotes develop in the fly gut then mature into epimastigotes which migrate to the salivary glands • Metacyclic trypomastigotes develop which are the infective stage occurs in the fly - Short, stumpy trypomastigote form, short flagellum the fly bites human and injects the MTs into the human, circulate into lymphatic and invade CNS, humans are primary revivor and so are cattle

T. cruzi - Routes of transmission

• T. cruzi - Routes of transmission • Reduvid bug, trans-placental, blood transfusion, organ transplant, lab accident, contaminated food or water (rare) must screen blood transfusions - Found only in the Americas • Endemic in Mexico, Central & South America • A few insect transmitted cases have occurred in Texas and California

Thick Blood Smears

• Thick Blood Smears - Used to screen for presence of parasites cant determine species - Allows for review of a large volume of blood, but doesn't maintain RBC morphology - Laked (RBCs lysed) before or during staining , so that the only structures on the slides are WBCs, platelets and parasites dime sized, 2-3 drops, must air dry, have to be able to read through - Preparation: • Spread a few drops of blood over a area about 2 cm in diameter - Should barely be able to see newsprint through the wet smear • Allow to air dry at room temperature takes around 6 hours • Do not fix with heat or methanol because we dont want RBCs to be fixed • Examine slide using both low power and oil objectives (observe the entire smear) giemsia- lyses during staining process, wright- has to have water sit on it for 10 min to let RBCs lyse

Helminths 2

• Transmission - Ingesting eggs - Ingesting larvae in an intermediate host - Direct larval penetration of the skin • Adults do not multiply in humans, so number of worms depends on number of eggs or larvae patient was exposed to - Worm burden is low tends to be asymptomatic, only see symptoms if worm burden is high • Presence and severity of symptoms is usually related to the worm burden folk remedies, tobacco, honey with water and vinegar, garlic and black walnut Anti helminth meds albendazole- prevents them from absorbing glucose ivermectin- paralyzes the worms praziquantel- concentrates calcium inside worms to cause cell death

Trypanosoma

• Trypanosoma - African sleeping sickness have west and east versions • Transmitted by tsetse flies - Chagas disease • Transmitted by Triatomine (reduvid) bugs or kissing bug • Can be transmitted through blood transfusion

Quality Control

• Updated procedure manuals • Controls for staining procedures • Record keeping - Centrifuge and ocular micrometer calibrations - Refrigerator and incubator temperatures • Parasitology reference book collection, images, permanent smears, and formalin preserved feces • Internal and external proficiency testing

Specimens for Urogenital Parasites

• Urine, Vaginal and Urethral Specimens - Eggs of Shistosoma haematobium and Enterobius vermicularis and trophs of Trichomonas vaginalis can be detected in urine sediment - T. vaginalis can also be detected in wet mounts of vaginal or urethral discharge more likely to see than urine sediment also called a wet prep

Modified Iron Hematoxylin Smear

• Used to distinguish among protozoan cysts and trophozoites • Background should be various shades of gray-blue • Protozoa should have medium-blue cytoplasm and dark blue-black nuclei

Helminths

• Worm like parasites • Classified according to their general external shape and the host organ they inhabit look at external and internal characteristics of eggs - Flukes (Trematodes) • Adults are leaf shaped flat worms - Tapeworms (Cestodes) • Adults are elongated, segmented flatworms - Roundworms (Nematodes) cylindrical like a worm

Microscopic Examination 3

• Zinc-Sulfate Floatation Method - Organisms are suspended at the top of a highdensity fluid • Zinc-sulfate has a specific gravity of 1.18 • Eggs and cysts will float on top of the solution because they have a lower specific gravity - Collected by placing a coverslip on the surface of the tube • This method will not detect operculated has a lid or heavy eggs some helminths 1 feces to 3-5 formalin, strain and centrifuge add ZS, resuspend until smooth and then recentrifuged, after 30 min organism will fall must collect on slide before that • Permanently Stained Smears - Provide contrast colors between the background debris and parasites • Allows better view of morphology and aids in recovery and ID of parasite - Specimen can be any fresh or preserved stool • Thin film of stool smeared onto slide • Dipped into Shaudinn's fixative before staining if stool is fresh - Observe a minimum of 300 fields using oil immersion dont use for helminths, only for protozoans

Apicomplexa: Babesia microti 2

• Zoonotic infection - White-footed mice are the usual reservoir, but also white-tailed deer and domestic animals • Transmitted by the Ixodes tick - Co-infection by Borrelia burgdorferi is common, lime disease makes symptoms much worse

Plasmodium Lifecycle

• pg 644- Includes both sexual reproduction (sporogony) and asexual reproduction (schizogony) • Sporogony stage in mosquitoes (definitive host) • Schizogony in humans has two phases, exoerythrocytic outside RBC and erythrocytic inside RBC - Exoerythrocytic phase • Sporozoites are injected by an infected female anopheles mosquito key • Move rapidly to the liver where they invade hepatocytes takes about 60 min - Exoerythrocytic phase • Maturation through the trophozoite and schizont phases leading to the formation and release of thousands of free merozoites into circulation • P. vivax and P. ovale can form hypnozoites (dormant bodies) which can resume development and produce disease at a much later time

Blood and tissue flagellates

•- Transmitted by vectors - Leishmania and Trypanosoma • Leishmania spp. - At least 20 species- more are being discovered regularly, taxonomy is in flux - Zoonotic infection- dogs and rodents are reservoirs, >30 species of sandflies are vectors just know that sandflies are vectors