Mycology, Introduction To Parasitology, Intestinal and Urogenital Protozoan Parasites, Blood &Tissue Protozoa, Platyhelminthes, Nematodes, Ectoparasites

Cryptosporidium parvum

Epidemiology: world wide, infects human and animals, millions of Crypto parasites shed in stools. Transmission by fecal- oral route, Approx. 748, 000 cases every year in US Biology: Sporozoan (coccidian) - exists in different forms, sexual and asexual reproduction. Acid fast oocyst (infective stage and diagnostic stage) Pathogenesis and Clinical disease: Adheres on the epithelial cells of the small intestine AIDS associated diarrhea, non bloody diarrhea, chronic, up to 50 stools Infects healthy persons as well.

Balantidium coli

Epidemiology: world wide, mostly tropical and subtropical, rare in US - infects animals - Risk factors: working with animals (pigs farm) and immune-compromised status fecal-oral route Outbreak associated with contaminated water supplies with pig feces. Biology: ciliate - cyst and trophozoite Trophozoite-covered with cillia (aid in motility), funnel like mouth (cytostome), has 2 nuclei - macro and micro nuclei, 50-200× 40-70 µm Cyst: refractile wall, single nucleus, 40-60µm (diameter)

Cystoisospora belli (Formerly, Isospora belli)

Epidemiology: worldwide, healthy as well as immunocompromised patients, Transm. by ingestion of contaminated food or water Least common of 3 intestinal coccidian Biology: sporozoan (coccidian), both asexual (schizogony) and sexual (gametogony) reproduction The least common of all 3 intestinal coccidian (CDC)

Formol Ether Concentration Technique

Ether adsorbs fecal debris & floats. Formalin fixes/ preserves the specimen.

Cyclospora cayetanensis

Epidemiology: Worldwide but common in tropical and subtropical regions; Also infects animals Travelers, indigenous persons living in developing countries and immuno- compromised (particularly, HIV infected) person Transmission: by ingestion of food/vegetables/fruits/water contaminated with sporulated oocyst, outbreaks in US since 1990s Biology: Single celled coccidian parasite, both sexual and asexual reproduction

Entamoeba histolytica

Epidemiology: world wide, highest incidence in tropical and subtropical regions; Transmission by fecal oral route; homosexual men; asymptomatic carriage common; prevalence in US: 1-2% Biology: Amoeba;trophozoite and cyst (maximum 4 nuclei)

Antihelminthic transport disruption

Benzimidazole - Mebendazole, Albendazole Broad spectrum anthelmintic Multiple paths: inhibits glucose transport and fumarate reductase, disrupts microtubules

Protozoan Parasites

1. Amoeba (Sarcodina)—motile via pseudopods, divide by binary fission, exist as metabolically active trophozoites and inactive, resistant cysts. 2. Flagellates (Mastigophora)—motile via long whip-like flagellae, divide by binary fission, exist in trophozoite and cyst forms, exceptions exist 3. Ciliates (Cilliata)—motile via short, brush like cilia, divide by binary fission (may conjugate), exist as trophozoites and cysts 4. Sporozoa (Coccidia)—reproduce by sexual &/or asexual means, exist in diverse cellular forms, trophozoites, sporozoites, oocysts, gametocytes, etc. May be intracellular

Anti-protozoal Drugs

1. Heavy metals: inhibits sylfhydryl group of enzymes and disrupt glycolysis Arsenicals: eg, Melarsoprol inhibits parasite pyruvate kinase (inactivates sulfhydryl groups) and causes decreased concentrations of ATP, pyruvate and phosphoenol pyruvate Antimonials: eg, Sodium stibogluconate 2. Inhibitors of DNA Replication Quinoline derivatives - Chloroquine, Primaquine Interfere with DNA replication and hemoglobin digestion Diamidines - Pentamidine: binds with DNA Nitroimidazoles - Metronidazole*- breaks DNA 3. Inhibitors of Folic Acid Biosynthesis: Inhibition of biosynthetic pathway Pyrimethamine inhibitis the enzyme dihydrofolate reductase (DHFR). Sulfonamides/Trimethoprim* 4. Inhibitor of pyruvate-ferridoxin oxidoreductase: Nitazoxanide: Inhibits anaerobic metabolism (protozoa and anaerobic bacteria) 5. Inhibitors of Protein Synthesis Paramomycins - Paramomycin (aminoglycoside) Tetracycline, clindamycin, spiramycin (macrolide) 6. Sesquiterpenes: Artemisinins, artemether React with heme moety causing free radical damage to parasite membranes Most effective antimalarial 7. Phosphocholine analogue: Miltefosine Act on key enzymes involved in metabolism of ether lipids present on the surface of parasite 8. Atovaquone-Proguanil (Malarone) Atovaquine inhibits electron transport chains of mitochondria of parasite Proguanil-antifolate (inhibits DHFR enzyme)

Zoonosis

A disease involving a parasite for which the normal host is an animal, and wherein man can also be infected Cryptosporidium, Trichinella etc are examples of zoonotic parasites that may be transmitted from animals to humans

Stool examination

A flotation media (for concentration) has a higher specific gravity than 1.20 to float many of the common parasite ova. Sheather's sugar solution has a specific gravity of 1.27 which is high enough to float any ova. Sedimentation use solutions of lower specific gravity Heavy eggs (Ascaris egg) Operculated eggs (Trematodes) Larvae (Strongyloides stercoralis) Cysts Floatation use a higher specific gravity than 1.20 Non Operculated eggs Trematodes (S. m.) Cestode Nematode(Hook worms) Cysts

Reservoir host

A host in which the infectious agent normally lives and multiplies. It acts as the source for transmission of infection to humans.

Parasite

A living organism requiring intimate prolonged contact with another living organism to meet some of its basic nutritional needs. It refers to eukaryotic pathogens.

Amoebic ulcer of the penis

A very rare clinical entity. We report a case of amoebic ulcer of the glans penis in a 47- year-old male homosexual, symptomatic with severe pain and foul-smelling hemopurulent discharge of acute onset. He had received systemic antibiotics like ciprofloxacin and azithromycin prior to presentation with no improvement. Diagnosis was confirmed by wet mount microscopic examination of the discharge. The patient responded well to a course of metronidazole.

Hyphal morphology (In molds)

A. Non septate or septate Non-septate or coenocytic : no separation of cells, mutinucleated Septate—cells separated by septa (cell wall) B. Dematiaceous (dark colored) or hyaline (colorless)

Infectious Mycoses

A. Superficial mycoses: superficial limited to outermost layers of the skin and hair. No inflammation B. Cutaneous mycoses: may extend into epidermis, invasive; hair and nail involvement C. Subcutaneous mycoses: involve dermis, subcutaneous tissues, muscle, and fascia - usually requires traumatic implantation D. Systemic mycoses: Start with localized infection such as in the lung (by inhalation); may disseminate to skin and other organs. E. Opportunistic mycoses: often normal flora or ever-present in environment, special conditions allow disease: -Suppression of normal flora: by antibacterial therapy -Diabetes -Immuno-suppression *HIV infection, use of immunosuppressive drugs etc.

Chlamydoconidia

Become larger than the hypha, are round and thick-walled , borne usually on the terminal end of the hyphae or along side.

Cell and tissue damage

A. Toxic products: Hydrolytic enzymes, cytolytic enzymes B. Mechanical Damage: blockage, pressure, migration of larva through tissue C. Immunopathology : Type I hypersensitivity (IgE mediated hypersensitivity): mast cell and eosinophil degranulation Type II and III type hypersensitivity: tissue damage and inflammation and scarring Type IV hypersensitivity: cell mediated; granuloma Type I: Helminthic infection, African trypanosomiasis Type II: Trypanosoma cruzi infection Type III: Malaria, Schistosomiasis, Trypanosomiasis Type IV: Leishmaniasis (Kala-azar), Schistosomiasis, Trypanosomiasis Competition for nutrition (malnutrition), cause loss of blood

Dermatophytide

An allergic rash caused by an inflammatory fungal infection (tinea) at a distant site. The rash is usually itchy like dermatitis, with bumps or blisters scattered on face, trunk and/or limbs.

Vector

An arthropod or other living carrier that transports a pathogenic organism from an infected to a non-infected host. Mechanical vectors: are non essential to the life cycle of the pathogen Biological vectors: serve as the site of some developmental events in the life cycle of the parasite

Evasion of immune system

Antigenic variation - within one life stage or due to different life stages (eg, African trypanosomiasis, Plasmodium, Babesia spp, Giardia) Encystation - uses the host response to wall itself off Antigenic mimicry: eg, Plasmodium spp, Trypanosomes, Schistosomes Antigen masking: Shistosoma mansoni Antigen shedding: Trichinella Masking/camoflage - uses discarded host antigens to hide by pasting them to their surface or molecular mimicry (eg, Hydatid cyst, filaria, Trypanosomes) Intracellular location Ig and/or complement proteases some produce DAF like molecule and destabilize C3b, others bind to C9 and prevent it from forming MAC (eg. Protease by Malarial parasite) Immunosuppression - diversion of the host immune response to ineffective immune response: Extracellular developmental stage secretes chemicals that down regulate a humoral (Th2) immune response Intracellular developmental stage secretes chemicals that down regulate a cell mediated (Th1) immune response Others suppress the immune system by producing antioxidant - interfere with macrophage killing

Fungal Cell Structure

Are Eukaryotic cells, unicellular or multicellular and have membrane bound organelles, nuclear membrane, cytoskeleton Fungal Cell membrane: Phospholipid bilayer like mammalian cells, but differs in the nature of sterol- they have ergosterol instead of cholesterol Site of action for antifungal drugs Amphotericin B and azole

Cystoisospora belli Life cycle

At the time of excretion in stool, the oocyst is immature and usually contains just one sporoblast (sometimes, two) . During further maturation after excretion, the sporoblast divides into two (the oocyst now contains two sporoblasts); the sporoblasts secrete a cyst wall, thus becoming sporocysts; and the sporocysts divide twice, resulting in four sporozoites per each of two sporocysts . Infection occurs by ingestion of mature (fully sporulated) oocysts: the sporocysts excyst in the small intestine and release their sporozoites, which invade the epithelial cells and initiate schizogony (asexual reproduction). Upon rupture of the schizonts, merozoites are released, which invade epithelial cells and continue the cycle of asexual multiplication. Trophozoites develop into schizonts, which contain multiple merozoites. After a minimum of one week, the sexual stage begins, with the development of male and female gametocytes . Fertilization results in the development of oocysts, which are excreted in the stool

Vegetative reproduction

Budding: Budding by constrictions results in Blastospores (Blastoconidia) If cells do not separate, they form pseudohyphae Fragmentation of hyphae: results in Arthrospores (Arthroconidia)

Balantidium coli: life cycle

Cysts are the parasite stage responsible for transmission of balantidiasis . The host most often acquires the cyst through ingestion of contaminated food or water . Following ingestion, excystation occurs in the small intestine, and the trophozoites colonize the large intestine . The trophozoites reside in the lumen of the large intestine of humans and animals, where they replicate by binary fission. Trophozoites undergo encystation to produce infective cysts. Some trophozoites invade the wall of the colon and multiply. Some return to the lumen and disintegrate. Mature cysts are passed with feces.

Plasmodium spp.

Causes Malaria Five species of medical importance P. falciparum (most serious form of malaria) P. vivax P. malariae P. ovale P. knowlesi: Infects primarily long-tailed & pig-tailed macaques human cases in Malaysia & other South east Asian countries)

Pathogenesis of Cystoisospora belli

Clinical disease: Asymptomatic or mild to severe GI disease (similar to Giardiasis); malabsorption Watery diarrhea with foul smelling stools

Pathogenesis of Trichomonas vaginalis

Clinical disease: Trichomoniasis - more common in female Female- found in urethra and vagina, male- in urethra and prostrate gland Female: Inflammation of the vagina and cervix with itching and burning. Vaginal discharge which is scant, frothy, greenish-yellowish Strawberry red cervix Males: usually asymptomatic as reservoir May cause urethritis with discharge

Molecular fungal diagnosis

Clinical sample: PCR: Amplification of Fungal DNA In situ hybridization: detection of fungal DNA in tissue biopsy Culture: use of DNA probes to identify fugal culture; PCR

Direct Light Microscopic Examination

Clinical sample: sputum, lung biopsy, skin scraping Wet mount or stained smears -Wet mount: KOH: Treat the sample with 10% KOH to clear debris; Stains -Gram stain: in suspected yeast infection, Candida may stain as G +. -Indian ink (negative staining): for detection of capsulated fungus -Calcofluor white stain: is fluorescent and binds to chitin -Other stains: Periodic acid Schiff (PAS), Gomori methanamine silver (GMS), Giemsa, Hematoxylin and eosin, immunofluorescent staining Determine Morphology: yeast or mold; hyphal structure, asexual spores

Fungal cell wall

Composed of Chitin,Glucan, Mannan and proteins Chitin: a polymer of N acetylglucosamine (provides rigidity) Major composition of fungal cell wall Glucan : a polymer of D-glucose - provide additional strength Mannan +protein: a polymer of the mannose sugar found linked to surface protein. May undergo antigenic variation, activate complement Glucan and Mannan = PAMP (induce innate immunity)

Entamoeba histolytica: life cycle

Cysts and trophozoites are passed in feces . Cysts are typically found in formed stool, whereas trophozoites are typically found in diarrheal stool. Infection by Entamoeba histolytica occurs by ingestion of mature cysts in fecally contaminated food, water, or hands. Excystation occurs in the small intestine and trophozoites are released, which migrate to the large intestine. The trophozoites multiply by binary fission and produce cysts, and both stages are passed in the feces. Because of the protection conferred by their walls, the cysts can survive days to weeks in the external environment and are responsible for transmission. Trophozoites passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive exposure to the gastric environment. In many cases, the trophozoites remain confined to the intestinal lumen (: noninvasive infection) of individuals who are asymptomatic carriers, passing cysts in their stool. In some patients the trophozoites invade the intestinal mucosa (: intestinal disease), or, through the bloodstream, extraintestinal sites such as the liver, brain, and lungs (: extraintestinal disease), with resultant pathologic manifestations. It has been established that the invasive and noninvasive forms represent two separate species, respectively E. histolytica and E. dispar. These two species are morphologically indistinguishable unless E. histolytica is observed with ingested red blood cells (erythrophagocystosis). Transmission can also occur through exposure to fecal matter during sexual contact.

Giardia lamblia: life cycle

Cysts are resistant forms and are responsible for transmission of giardiasis. Both cysts and trophozoites can be found in the feces (diagnostic stages) . The cysts are hardy and can survive several months in cold water. Infection occurs by the ingestion of cysts in contaminated water, food, or by the fecal-oral route (hands or fomites) . In the small intestine, excystation releases trophozoites (each cyst produces two trophozoites). Trophozoites multiply by longitudinal binary fission, remaining in the lumen of the proximal small bowel where they can be free or attached to the mucosa by a ventral sucking disk. Encystation occurs as the parasites transit toward the colon. The cyst is the stage found most commonly in nondiarrheal feces . Because the cysts are infectious when passed in the stool or shortly afterward, person-to-person transmission is possible. While animals are infected with Giardia, their importance as a reservoir is unclear.

Diagnosis of Trichomonas vaginalis

Demonstration of characteristic trophozoite in vaginal and urethral discharge; (unstained smears or Giemsa, papanicolaou stained smear) On Microscopy of wet preparation, T. vaginalis, which is larger than Polymorphonuclear leukocytes but smaller than an epithelial cell, it is recognized by its usually rapidly moving flagella, the rippling movement of undulating membrane and the jerky movement of the organism.

Diagnosis of Balantidium coli

Demonstration of cyst and trophozoite in feces

Diagnosis of Giardia lamblia

Demonstration of cyst or trophozoites Several stool samples may be necessary String test/ Entero test Usually a stool sample is tested first. A string test is done if the stool sample is negative. String test: a string with a weighted gelatin capsule is swallowed. Four hours later, it is pulled back out. Any bile, blood, or mucus (from small intestine) attached to the string is examined under the microscope for cells and parasites or parasite eggs.

Diagnosis of Cystoisospora belli

Demonstration of oocyst (Approx.25 × 15µm) Concentrated stool specimens and Iodine mount or Modified Acid fast stained smears

Laboratory Diagnosis of Cyclospora cayetanensis

Demonstration of oocysts by Modified Ziehl-Neelsen (acid-fast) staining of formol-ether concentrated stool samples The oocysts are variably acid-fast. Demonstration of autofluorescence: The oocysts are refractile spheres which exhibit blue autofluorescence under ultraviolet light. Molecular: PCR

Amoebic Liver Abscess

Diagnostic aspiration: Aspirated pus examined for the presence of trophozoites (Not cysts!!!!) Or, Liver biopsy Along with examination of stool, Serological tests Radiological examination PCR and DNA probe assay

Fungal Reproduction

I. Asexual A. Vegetative reproduction By budding (in yeast) or fragmentation of hyphae B. Formation of asexual spores: Anamorph Several types: important in identification II. Sexual: Formation of sexual spores: Teleomorph Not used for identification, but for taxonomy

Treatment of Trichomonas vaginalis

Drug of choice: Metronidazole Prevention and control Safe sexual practices Personal hygiene Avoidance of sharing toilet articles

Treatment, Prevention and Control

Drug of choice: Tetracycline Prevention and control similar to amoebiasis When traveling to endemic tropical countries, Balantidium coli infection can be prevented by following good hygiene practices. All fruits and vegetables should be with clean water when preparing or eating them, even if they have a removable skin.

Treatment of Cystoisospora belli

Drug of choice: Trimethoprim-Sulfamethoxazole Prevention and Control: Maintaining personal hygiene and sanitary conditions

Malarial Infection of RBC

During the erythrocytic stage of the parasite's life cycle, it uses intracellular hemoglobin as a food source. The protein is broken down into peptides, and the heme group is released and detoxified in the form of hemozoin Maurer clefts/dots: finely granular precipitates or irregular cytoplasmic particles that usually occur diffusely in red blood cells infectedwith the trophozoites of Plasmodium falciparum. They are membrane-limited vacuoles or sack-like structures in the cytosol of the erythrocyte, formed early after invasion by the parasite, visible as blue dots in Giemsa-stained blood smears. Their origin and functions remain still unclear.

Amoebiasis (amoebic dysentery)

Entamoeba histolytica Intestinal (large intestine) - IP: 4-5 days: dysentery (bloody diarrhea), abdominal pain, cramping; Severe case: numerous bloody stools/day Asymptomatic carriage Extra intestinal: liver abscess: seen approx. 5% of intestinal inf. pain and tenderness in right hypochondrium, fever, hepatomegaly (lower border of the liver palpable), transmission to other regions Spleen and cutaneous amoebiasis brain abscess Genital abscess - (in homosexual men)

Laboratory Diagnosis of Intestinal Amoebiasis

Examination of stool: -Macroscopic examination: offensive, darkbrown semi-fluid stool mixed with blood and mucous -Microscopic examination: Demonstration of E. histolytica-Cysts/trophozoites Presence of Cellular exudate and Charcol-Leyden crystals Charcot-Leyden crystals are slender and pointed at both ends. They consist of Lysophospholipase (an enzyme synthesized by eosinophils) and are produced from the breakdown of eosinophils PCR and DNA probe assay

Life cycle

For survival and reproduction, many parasites evolve through a number of morphologic stages and several environments or different hosts. The sequence of morphologic and environmental stages is referred to as the life cycle.

Immunological fungal diagnosis

For systemic mycosis Dermal hypersensitivity testing (like tuberculin test) Serology: Detection of antibody (4x increase of paired sera) Detection of fungal antigens in patient's sample

Fungal Sexual Reproduction

Formation of sexual spores (Teleomorph) 4 classes based on sexual spore formation Zygomycetes: have non septate hyphae, produce zygospores as sexual spores (They also produce sporangiospores as asexual spores) Ascomycetes: Include both yeasts and filamentous fungi (Septate), form ascospores (sexual spores) Basidiomycetes: Basidiospores on a basidium or base Deuteromycetes: Also called as Fungi imperfecti Provisional group whose sexual phase not identified Contains most fungi of medical importance

Sporangiospores

Formed in a sac called a sporangium, easily broken to free the spores

Laboratory Diagnosis of mycotic disease

Four approaches 1. Direct Light Microscopic Examination 2. Culture 3. Immunological tests/Serology 4. Molecular: eg, DNA probe tests

Fungi as Infectious Agents

Fungi are widely distributed in nature Only few are associated with disease True/primary pathogens can cause disease in a healthy, immunocompetent host Opportunistic pathogens are fungi with weak/nonexistent virulence or invasiveness, cause disease in immunocompromised host Increase in incidence - 10% of all hospital acquired infections

Giardia lamblia

G. duodenalis/G. intestinalis Epidemiology: world wide; In US, Giardia infection is the most common intestinal parasitic disease Also Infects other animals (beavers and muskrats) Transmission: fecal oral- mostly via contaminated water (such as streams, rivers); food and fingers; oral-anal sexual Risk factors-travel to known endemic areas, consumption of inadequately treated water, day care centers and oral-anal sexual practices Biology: flagellate - cyst and trophozoite Cyst: oval, 4 nuclei, axostyle/axoneme Trophozoite: Tennis/badminton racket appearance, two nuclei, 4 pairs of flagella, axoneme, sucking disc on ventral surface, 'Falling leaf' motility

Pathogenesis of Giardia lamblia

Giardiasis Adhere to the wall of small intestine Malabsorption of food (fats and fat-soluble vitamins) Intestinal: non-bloody diarrhea: bulky, greasy stool/steatorrhea (Incubation Period: average of 10 days)

Fungal Morphology

I. Yeasts—oval, unicellular, reproduce by budding (blastoconidia) eg, Cryptococcus neoformans II. Yeast like: yeasts but form pseudohyphae (chain of elongated yeast cells, not true hyphae) during invasion of tissue eg, Candida albicans III. Mold form: thread-like tubular multicellular hyphae, colonies have cottony or velvety surface. Intertwining mass of hyphae is known as mycelium eg, Aspergillus spp. IV. Dimorphic: exist in 2 forms-yeast (at 37 0C) and mold (at 25 0C) interconversion between them influenced by temperature - Thermal dimorphism. Eg, Histoplasma capsulatum

Cytoadherence

In P. falciparum infections, membrane protuberances appear on the erythrocyte's surface 12-15 h after the cell's invasion. These "knobs" extrude a high-molecular-weight, antigenically variant, strain-specific erythrocyte membrane adhesive protein (PfEMP1) that mediates attachment to receptors on venular and capillary endothelium—an event termed cytoadherence.

Trichomonas vaginalis life cycle

In the United States, an estimated 3.7 million people have the infection, but only about 30% develop any symptoms of trichomoniasis. Infection is more common in women than in men Female- found in urethra and vagina, male- in urethra and prostrate gland Sexual transmission is the primary mode of transmission.

Major Protozoan Parasites

Intestinal -Entamoeba histolytica -Giardia lamblia -Cryptosporidium parvum -Cyclospora cayetanensis* -Cystoisospora belli * -Balantidium coli * mostly in immunocompromised host Urogenital -Trichomonas vaginalis Blood and tissue -Plasmodium spp. -Babesia spp. -Toxoplasma gondii -Leishmania spp. -Trypanosoma spp. -Naegleria fowleri -Acanthamoeba castellani

Pathogenesis of Plasmodium

Intracellular parasite, infects red blood cells and hepatocytes, dormancy in hepatocytes (except P. falciparum) P. falciparum: The processes of cytoadherence, rosetting, and agglutination are central to the pathogenesis of falciparum malaria Sequestration of RBCs containing mature forms of the parasite in vital organs (particularly the brain) interfere with microcirculatory flow and metabolism Clinical disease: Malaria : Incubation period: approx. 2 wks after bite. Fever, chills and sweating, shows periodic cycle. Febrile paroxysm synchronizes with erythrocytic schizogony, Types of fever: A. Tertian fever (with 48 hr cycle): recurs every 3rd d B. Quartan fever (with 72 hr cycle): recurs every 4th d C. Quotidian fever: recurs every 24 hrs Hepatosplenomegaly and anemia.

Lab Diagnosis of Cryptosporidium parvum

Lab Diagnosis: Demonstration of oocyst in feces; unstained, acid fast or fluorescent staining

Plasmodium: Life Cycle

Life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host . Sporozoites infect liver cells and mature into schizonts , which rupture and release merozoites . (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later). After this initial replication in the liver (exo-erythrocytic schizogony ), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony) . Merozoites infect red blood cells . The ring stage trophozoites mature into schizonts, which rupture releasing merozoites . Some parasites differentiate into sexual erythrocytic stages (gametocytes) . Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal . The parasites' multiplication in the mosquito is known as the sporogonic cycle . While in the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes . The zygotes in turn become motile and elongated (ookinetes) which invade the midgut wall of the mosquito where they develop into oocysts . The oocysts grow, rupture, and release sporozoites , which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle.

Stool/urine sample

Macroscopic examination Microscopic examination Wet mount Permanent stains Stool concentrates Demonstrate: Cyst, trophozoite, egg, larvae, adult Culture

Entamoeba histolytica Pathogenesis

Major virulence factors are Lectin/adherence protein Release of cytoxins and hydrolytic enzymes -Cytotoxin/Amoebapores (pore forming peptide): create pores in the target cell -Cysteine protease: (Thiol protease): degrades the extracellular matrix

Black water fever (dark urine)

Manifestation of falciparum malaria in previously infected subjects. Intra vascular hemolysis, fever, hemoglobinuria, Can result in acute renal failure and death In majority cases, parasites not detected in periph. blood.

Conidia

May arise directly from hyphae or from specialised stalk-like structures - conidiophore

Blood &Tissue Protozoa

Medically Important: Plasmodium spp. Babesia microti Toxoplasma gondii Leishmania spp. Trypanosoma spp. Naegleria fowleri Acanthamoeba castellani

Treatment for Entamoeba histolytica

Metronidazole followed by iodoquinol (quinoline derivative) Asymptomatic carriage: iodoquinol Prevention and Control Education Public and personal hygiene Use of boiled water/Protection of all food and drink from contamination by flies, cockroaches and rats Avoidance of raw vegetables and fruits Amoebicidal drugs: Tissue amoebicides (Trophozoites): Emetine, Dehydroemetine Luminal (Both trophozoites and cysts): Iodoquinol, Diloxanide Both Tissue and luminal: Metronidazole, Niridazole

Treatment and prevention of Giardia lamblia

Metronidazole or nitazoxanide with furazolidone (Nitrofuran antimicrobial, works by crosslinking of DNA) Prevention and control: Personal and public hygiene Avoidance of contaminated water and food Boiling or filtration (water from streams/lakes) Proper filtration system in municipal water supplies (since cysts are resistant to standard chlorination) Avoidance of high-risk sexual behaviour

Blood and tissue samples

Microscopic examination Blood, CSF, biopsy Serologic examination Antibody response Antigen detection Nucleic acid hybridization PCR Detection - In situ Culture Imaging; detect presence of parasites in tissues Xenodiagnosis Blood differential cell count = increased eosinophils in parasitic infection

Fungal Growth

Most fungi are aerobic, some may be facultative anaerobic Are heterotrophic - require organic matter for growth Most grow best at room temperature 20 -40 0C Fungi will grow well on slightly acidic media, slightly drier, higher osmotic pressure where bacteria do not Common culture media - Sabouraud dextrose agar (SDA) Selective for fungi: pH 5.6, osmotic pressure: Does not support bacteria Cycloheximide may be added to SDA to suppress fast growing non pathogenic fungi -To favor growth of pathogenic one in a mixture of same. Thermal dimorphism : Yeast at 37 0C and mycelial form at room temp (25 0C) Fungi can withstand higher osmotic pressures than most bacteria

Cerebral malaria

Most severe neurological complication of infection with Plasmodium falciparum malaria; Coma; Only young ring forms and occasionally gametocytes peripheral blood Mortality high, surviving patients sustain brain injury- long-term neuro-cognitive impairments.

Hypersensitivity Mycotic Diseases

Non Infectious Airborne spores, hyphal fragments; Id reaction

Pathogenesis of Fungi

Source Exogenous: Environmental exposure, spore or hyphal form can be infectious Some are geographically restricted Endogenous: Overgrowth of normal flora Route of Entry: respiratory, cutaneous, or through mucus membranes Virulence factors: Include -Cell wall components are inflammatory stimulants -Adhesion factors -Capsules -Survival inside macrophages -Hydrolytic enzymes (proteases, phospholipases) -Thermal dimorphism: ability to grow at 37oC -Mycotoxins: toxic metabolites produced in the environment; associated with mycotoxicosis Some fungi produce melanin which interfere with the oxidative killing inside macrophages.

Host

Organism harboring a parasite. Definitive host: Animal harboring the adult or sexually mature stage of the parasite. Intermediate host: Animal in which development occurs but in which adulthood is not reached.

Transmission of Parasitic Disease

Source: Exogenous (from out side) Mode of transmission Ingestion: Contaminated water or food with human/animal waste Raw or undercooked beef/pork/fish Direct penetration: Arthropod borne, larval-directed skin penetration Transplacental Sexual contact

Major differences between C. cayetanensis and C. parvum

Size difference — C. parvum is smaller; Differing results from modified acid-fast staining -C. parvum has consistent red staining; and Autofluorescence under UV light — C. cayetanensis does, C. parvum does not.

Antifungal Drugs

Polyenes : Amphotericin B, Nystatin Affects integrity of plasma membrane: Inserts in fungal membrane next to ergosterol; causes pore formation, ion leakage Azoles : Eg. Ketoconazole, Fluconazole inhibit plasma membrane synthesis: by interfering with ergosterol synthesis Echinocandins : eg. Caspofungin inhibits cell wall synthesis - interfere with glucan synthesis Nikkomycins: Inhibit cell wall synthesis: interfere with chitin synthesis Flucytosine : Inhibits nucleic acid synthesis Griseofulvin: : Inhibits cell division: interfere with microtubules (disruption)

Replication

Protozoans: multiply inside the human body Intracellular or extracellular - depending on organism Helminths: most do not replicate inside the human body

Antihelminthic Inhibition of Neuromuscular Action

Pyrazinoisoquinoline: Praziquantel Calcium agonist, tetanic muscular contraction as well as destruction of the tegument Tetrahydropyrimidine: Pyrentel pamoate- cholinergic agonist Piperazines - Diethylcarbamazine (DEC): stimulate cholinergic recep. Muscular paralysis, enhances adherence of leucocyte on parasite Avermectins - Ivermectin Interacts with chloride channel, blocks neuromuscular action Phenols-Niclosamide Acts by uncoupling of oxidative phosphorylation in mitochondria-loss of helminth ATP-Immobilization

Pathogenesis of Balantidium coli

Release of proteolytic and cytotoxic sustances Causes invasion and ulceration Extraintestinal-rare Clinical disease: Abdominal pain and tenderness, tenesmus, Bloody diarrhea/dysentery similar to that of Entamoeba histolytica

Pathogenesis of Cyclospora cayetanensis

Rupture of epithelial cells during release of oocyst Incubation Period: an average of 7 days Watery diarrhea (most common), loss of appetite, weight loss, cramping, bloating, increased gas, nausea, fatigue If not treated, persistent watery diarrhea lasting over several days Typically, patients who come in with a persistent watery diarrhea lasting over several days may be suspected of harboring the disease, especially if they have traveled to a region where the protozoan is endemic.

Fungal Culture

Sabouraud dextrose agar (SDA): a common culture media Slightly acidic, may contain antibacterial drugs to suppress bacterial growth Two cultures - at 25 oC and 37 oC - detect thermal dimorphism Not reported negative until 4 weeks Gross colony appearance such as color, texture etc.; and Pigmentation Microscopic examination: examine hyphal and spore structures Lactophenol Cotton Blue (LPCB) mount-commonly used Components of LPCB stain: Lactic acid: preservative Phenol: disinfectant Cotton blue: stain Glycerine: hygroscopic agent

Laboratory Diagnosis of parasites

Samples: based on suspicion of organ invasion Stool samples: a series of 3 specimens on alternate days (within 10 days) Urine sample Blood and tissue etc.

Treatment of Cryptosporidium parvum

Self limiting in healthy. No effective treatment, Immunocompromised may use paromomycin, nitazoxanide Prevention and control: Improved personal hygiene and sanitation, purification of water supply including filtration (since cysts are resistant to standard chlorination)

Protozoa

Single cells; eukaryotic cell structure Surrounded by either a plasma membrane alone or with an exterior flexible pellicle Extracellular and intracellular infections May have two or more forms/stages Trophozoite is the motile, metabolically active, replicating form Cyst is the dormant, non-replicating form that is adapted for survival in the environment. If it does not have a cyst form, it must be transmitted via a vector or direct contact

Morphology of Plasmodium spp.

Sporozoan, exists in different forms (sexual and asexual reproduction). Sporozoites : infective to liver cells Trophozoites: early stages in liver/RBC Schizont - mature malarial parasite, contains many merozoites. Merozoites: infective to RBC Gametocyte: inside RBC

Cryptosporidium parvum Life cycle

Stages: several forms Oocyst (with sporozoites inside): inactive: Infective and diagnostic stage: Trophozoite; actively dividing Merozoite Gametes: micro and macro gamonts Gamete fusion - zygote Oocyst

Hyphae

The branching, threadlike tubes that make up the bodies of multicellular fungi

Intestinal Amoeba differential diagnosis

The presence of commensals amoebas in the intestine may make microscopic diagnosis difficult. E. histolytica can be distinguished from the other amoebas by trophozoite and cyst morphology. Number of nuclei in the cyst - maximum 4. Shape of the nuclei - central karyosome. Presence of ingested RBC in trophozoite are characteristic of E. histolytica

Formation of asexual spores (Anamorph)

The shape, color and arrangement of asexual spores/spore forming structures are used in the identification of fungi Two basic forms: a. Conidia-naked spores, no sac, most common form ; different forms Eg, Chlamydoconidia, Microconidia, Macroconidia etc. b. Sporangiospores - contained in a sac like structure

Toxin mediated diseases

Toxin interrupt physiological functions. A. Mycotoxicoses: Results from ingestions of mycotoxins produced by fungi on food Ergot Alkaloids-by mold growing in grains (Claviceps purpurea) Causes ergotism- Hallucinations, GI-upset, dry gangrene and painful burning sensation: outbreaks Aflatoxin - produced by Apergillus flavus in moldy grains, peanuts carcinogenic toxin - induce mutation on p53 (growth suppressor gene) - causes liver cancer B. Mycetismus (Mycetism/Mushroom poisoning)results from eating poisonous mushrooms (Amanita spp) Amanitin/Phalloidin: Toxin resistant to heating - cooking does not inactivate May cause severe/fatal liver and kidney failure

Treatment of Cyclospora cayetanensis

Trimethoprim/sulfamethoxazole Prevention and Control Increased sanitation and hygiene practices Warn travelers not to visit regions where the coccidian parasites are endemic Treatment with chlorine or iodine is unlikely to kill Cyclospora oocysts. No vaccine for cyclosporiasis is available

Epidemiology of Malaria

Tropical and subtropical, human to human via a vector- mosquito (Anopheles spp), by blood transfusions/organ transplant, also by IV drug use Malaria is one of the most severe public health problems worldwide. It is a leading cause of death and disease in many developing countries 1-5 billion febrile episodes and 1-3 million deaths annually. Approx. 1,500-2,000 cases every year in US Airport malaria: In Palm Beach County, Florida, during the summer of 2003, a cluster of eight cases of malaria was detected

Oocysts of Cyclospora cayetanensis

Twice as large in comparison with C. parvum and are not sporulated (do not contain sporocysts) upon excretion

Fungal Diseases

Two categories: 1. Infectious : Mycosis (plural-Mycoses) 2. Non infectious most common site is the skin, - dermatophytes -candida

Metazoa (helminths)

Two groups 1. Platyhelminthes - flat worms: two forms- a) Cestodes: segmented body, (proglottids), no alimentary tract, head has suckers &/or hooks for attachment, hermaphroditic b) Trematodes: leaf shaped, oral/ventral suckers, blind alimentary tract, usually hermaphroditic 2. Nemathelminthes/Nematodes—round worms, long with true alimentary tract, oral attachment device possible, produce eggs/ larvae, separate sexes

Adherence

Two types: specific or nonspecific Specific: molecular recognition Duffy antigen is binding site for Plasmodium vivax Non specific: Mechanical "Hooks" in hookworms nonspecific

Trichomonas vaginalis

Very common agent of STDs Epidemiology: world wide, prevalence in developed countries-5-20% women and 2-10% men; In US: 3.7 million people infected, Transmission: sexual transmission sharing shower materials, through mother's infected birth canal- infection of infants Biology: flagellate; Anerobic exists as trophozoite only (no cysts!!!) one nucleus, four flagella and undulating membrane, a locomotory organelle of certain flagellate (trypanosome and trichomonad) parasites, consisting of a finlike extension of the limiting membrane with the flagellar sheath; wavelike rippling of the undulating membrane produces a characteristic movement. Costa: Ribbon like supporting structure at the base of undulating membrane

Cyclospora cayetanensis : Life cycle

When freshly passed in stools, the oocyst is not infective (thus, direct fecal-oral transmission cannot occur; this differentiates Cyclospora from another important coccidian parasite, Cryptosporidium). In the environment , sporulation occurs after days or weeks at temperatures between 22°C to 32°C, resulting in division of the sporont into two sporocysts, each containing two elongate sporozoites . Fresh produce and water can serve as vehicles for transmission and the sporulated oocysts are ingested (in contaminated food or water) . The oocysts excyst in the gastrointestinal tract, freeing the sporozoites which invade the epithelial cells of the small intestine. Inside the cells they undergo asexual multiplication and sexual development to mature into oocysts, which will be shed in stools. The potential mechanisms of contamination of food and water are still under investigation.

Fungal Colony Morphology

Yeast colony: opaque, compact, mucoid/dry, bacteria like, alcoholic odor, grow well at room temp in 24 hrs Molds colony : Hairy, cottony, velvety texture, may be colored (dematiaceous-due to pigment production). Vegetative hyphae: extend into growth medium, absorb nutrition Aerial (reproductive) hyphae: extend from the agar surface and often have spores- useful in identifying the mold