2. PROTOZOAN INFECTIONS - PARASITOLOGY.pdf

PROTOZOAN INFECTIONS Vicente Belizario, Jr., Winifreda de Leon 2013, University of the Philippines Manila, Medical Parasitology in the Philippines REMAUR S. HERRERA, DVM, MD INTESTINAL AMEBAE  Seven species of amebae occur in humans  They are mainly differentiated on the basis of structure and...

PROTOZOAN INFECTIONS Vicente Belizario, Jr., Winifreda de Leon 2013, University of the Philippines Manila, Medical Parasitology in the Philippines REMAUR S. HERRERA, DVM, MD INTESTINAL AMEBAE  Seven species of amebae occur in humans  They are mainly differentiated on the basis of structure and size  Pathogenic Entamoeba histolytica  Commensals E. dispar, E. moshkovskii, E. hartmanni, E. coli, Endolimax nana, and Iodamoeba butschlii.  Entamoeba polecki is an intestinal ameba of pigs and monkeys that has been occasionally detected in humans, and is a probable cause of diarrhea. ENTAMOEBA HISTOLYTICA Nomenclature  Subphylum Sarcodina  Superclass Rhizopoda  Class Lobosea  Order Amoebida  Family Entamoebidae  Genus Entamoeba ENTAMOEBA HISTOLYTICA  The members of this genus are characterized by:  Having a vesicular nucleus  A centrally (or near central) located small karyosome  Varying numbers of chromatin granules adhering to the nuclear membrane ENTAMOEBA HISTOLYTICA  Nuclear and other morphologic differences distinguish the species of Entamoeba except E. histolytica, E. dispar, and E. moshkovskii (three said species are morphologically identical and of the same size)  E. hartmanni, formerly referred to as “small race” of E. histolytica, is differentiated primarily on the basis of size. ENTAMOEBA HISTOLYTICA Parasite Biology  Is a pseudopod-forming non-flagellated protozoan parasite.  It is the most invasive of the Entamoeba parasites and the only member of the family to cause colitis and liver abscess.  The life cycle of E. histolytica consists of two stages: an infective cyst and an invasive trophozoite form.  The quadrinucleate cyst is resistant to gastric acidity and desiccation, and can survive in a moist environment for several weeks.  No host other than humans is implicated in the life cycle, although natural infection of primates has been reported. ENTAMOEBA HISTOLYTICA Parasite Biology  Mode of Transmission  Ingestion of cysts from fecally-contaminated material.  Venereal transmission through fecal-oral contact  Direct colonic inoculation through contaminated enema equipment. ENTAMOEBA HISTOLYTICA Parasite Biology  Excystation occurs in the small or large bowel, where a cyst undergoes nuclear followed by cytoplasmic division to form eight trophozoites. ENTAMOEBA HISTOLYTICA Parasite Biology  E. histolytica trophozoites  Highly motile and possess pseudopodia.  They vary in size from 12 to 60 μm in diameter (about 20 μm in average).  Microscopic examination of fully-passed stool specimens reveals the characteristic progressive and directional movement of trophozoites, with pseudopodia as locomotory organelles.  The hyaline pseudopodium is formed when the clear, glasslike ectoplasm, or outer layer is extruded, and the granular endoplasm flows into it. ENTAMOEBA HISTOLYTICA Parasite Biology  E. histolytica trophozoites  Ingested red blood cells are observed as pale, greenish, refractile bodies in the cytoplasm of the ameba.  Trophozoites have the ability to colonize and/or invade the large bowel  E. histolytica trophozoites multiply by binary fission.  They encyst producing uninucleate cysts, which then undergo two successive nuclear divisions to form the characteristic quadrinucleate cysts. ENTAMOEBA HISTOLYTICA Parasite Biology Entamoeba histolytica trophozoite (From World Health Organization. Bench Aids for the Diagnosis of Intestinal Parasites. Geneva: World Health Organization; 1994) ENTAMOEBA HISTOLYTICA Parasite Biology  E. histolytica cysts  Usually spherical, and the size may vary from 10 to 20 μm.  They are characterized by a highly refractile hyaline cyst wall, one to four nuclei, and rod-shaped (or cigar-shaped) chromatoidal bars.  Unlike trophozoites, cysts are never found within invaded tissues of the GIT. ENTAMOEBA HISTOLYTICA Parasite Biology Entamoeba histolytica quadrinucleate cyst (From Entamoeba histolytica cyst (Courtesy World Health Organization. Bench Aids for the of the Department of Parasitology, UP- Diagnosis of Intestinal Parasites. Geneva: World CPH) Health Organization; 1994) ENTAMOEBA HISTOLYTICA Parasite Biology  E. histolytica is a eukaryotic organism but has several unusual features, including the lack of organelles that morphologically resemble mitochondria.  There is no rough endoplasmic reticulum or Golgi apparatus, although cell surface and secreted proteins contain signal sequences, and tunicamycin inhibits protein glycosylation.  Ribosomes form aggregated crystalline arrays in the cytoplasm of the trophozoite. ENTAMOEBA HISTOLYTICA Parasite Biology  Some differences in biochemical pathways from higher eukaryotes include the lack of glutathione metabolism, the use of pyrophosphate instead of ATP at several steps in glycolysis, and the inability to synthesize purine nucleotides de novo.  Glucose is actively transported into the cytoplasm, where the end products of carbohydrate metabolism are ethanol, carbon dioxide, and under aerobic conditions, acetate. ENTAMOEBA HISTOLYTICA ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  The proposed mechanisms for virulence are:  Production of enzymes or other cytotoxic substances  Contact-dependent cell killing  Cytophagocytosis ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  In vitro, amebic killing of target cultivated mammalian cells involve receptor-mediated adherence of ameba to target cells, amebic cytolysis of target cells, and amebic phagocytosis of killed or viable target cells.  E. histolytica trophozoites adhere to the colonic mucosa through a galactose- inhibitable adherence lectin (Gal lectin).  Amebae kill mucosal cells by activation of their caspase-3, leading to their apoptotic death engulfment. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  Amebic colitis  Clinically presents as gradual onset of abdominal pain and diarrhea with or without blood and mucus in the stools.  Fever is not common and it occurs only in one third of patients.  Although some patients may only have intermittent diarrhea alternating with constipation, children may develop fulminant colitis with severe bloody diarrhea, fever, and abdominal pain. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  Ameboma  Occurs in less than 1% of intestinal infections  It clinically presents as a mass-like lesion with abdominal pain and a history of dysentery.  It can be mistaken for carcinoma. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  Amebic liver abscess (ALA)  The most common extra-intestinal form of amebiasis.  The cardinal manifestations of ALA are fever and right upper quadrant (RUQ) pain (most frequent complaints, particularly in acute cases (2 weeks duration) is found in older patients and it involves wasting with significant weight loss rather than fever.  Only 30% of ALA cases have concurrent diarrhea.  Mortality in uncomplicated ALA is less than 1%. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations Onset of infection  The onset of amebic colitis may be sudden after an incubation period of 8 to 10 days, or after a long period of asymptomatic cyst carrier state.  ALA may have acute presentation of less than 2 weeks duration or a chronic one of more than 2 weeks duration. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations Complications  The most serious complication of amebic colitis is perforation and secondary bacterial peritonitis.  Colonic perforation occurs in 60% of fulminant colitis cases.  In ALA, the most serious complications are rupture into the pericardium with a mortality rate of 70%, rupture into the pleura with mortality of 15 to 30%, and super infection.  Intraperitoneal rupture, which occurs in 2 to 7.5% of cases, is the second most common complication.  It is not as serious as colonic perforation because ALA is sterile. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations Complications  Secondary amebic meningoencephalitis occurs in 1 to 2%, and it should be considered in cases of amebiasis with abnormal mental status.  Renal involvement caused by extension of ALA or retroperitoneal colonic perforation is rare.  Genital involvement is caused by fistulae from ALA and colitis or primary infection through sexual transmission. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  Natural or innate immunity to E. histolytica in the intestines involves  Mucin inhibition of amebic attachment to the underlying mucosal cells.  Complement-mediated killing of trophozoites (systemic circulation)  Acquired immunity primarily involves  Cell-mediated responses and humoral responses ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  Activated T-cells kill E. histolytica by:  Directly lysing trophozoites in a contact-dependent process  Producing cytokines which activate macrophages and other effector cells (neutrophils and eosinophils)  Providing helper effect for B-cell antibody production. ENTAMOEBA HISTOLYTICA Pathogenesis and Clinical Manifestations  Cytokine studies revealed that interferon (IFN) and interleukin (IL-2) may have a role in activating macrophages for amebicidal activity.  Activated macrophages produce nitric oxide (NO) which was lethal to trophozoites.  Tumor necrosis factor (TNF) was shown to stimulate NO production.  Studies have revealed that the principal antibody-dependent cell cytotoxicity (ADCC) did not work against amebae. ENTAMOEBA HISTOLYTICA Differential Diagnosis  Acute amebic colitis should be differentiated from:  Bacillary dysentery caused by Shigella, Salmonella, Campylobacter, Yersinia, and enteroinvasive Escherichia coli.  Inflammatory bowel disease ENTAMOEBA HISTOLYTICA Differential Diagnosis Comparison of bacillary and amebic dysentery BACILLARY DYSENTERY AMEBIC DYSENTERY May be epidemic Seldom epidemic Acute onset Gradual onset Prodromal fever and malaise common No prodromal features Vomiting common No vomiting Patient prostrate Patient usually ambulant Watery, bloody diarrhea Bloody diarrhea ENTAMOEBA HISTOLYTICA Differential Diagnosis Comparison of bacillary and amebic dysentery BACILLARY DYSENTERY AMEBIC DYSENTERY Odorless stool Fishy odor stool Stool microscopy: few bacilli, red cells, trophozoites Stool microscopy: numerous bacilli, pus cells, with ingested red blood cells, Charcot-Leyden macrophages, red cells, no Charcot-Leyden crystals crystals Abdominal cramps common and severe Mild abdominal cramps Tenesmus common Tenesmus uncommon Natural history: spontaneous recovery in a few days, Natural history: lasts for weeks; dysentery returns weeks or more; no relapse after remission; infection persists for years ENTAMOEBA HISTOLYTICA Differential Diagnosis  ALA should be differentiated from:  Pyogenic liver abscess, tuberculosis of the liver, and hepatic carcinoma.  Genital amebiasis should be differentiated from:  Carcinoma, tuberculosis, chancroid, and lymphogranuloma venereum. ENTAMOEBA HISTOLYTICA Diagnosis  Standard method of diagnosis is microscopic detection of the trophozoites and cysts in stool specimens.  Ideally, a minimum of three stool specimens collected on different days should be examined.  For detection of trophozoites, fresh stool specimens should be examined within 30 minutes from defecation.  The detection of E. histolytica trophozoites with ingested red blood cells is diagnostic of amebiasis. ENTAMOEBA HISTOLYTICA Diagnosis  Unidirectional movement is characteristic of E. histolytica.  Using saline and methylene blue, Entamoeba species will stain blue, thus, differentiating them from white blood cells.  Using saline and iodine, the nucleus and karyosome can be observed to differentiate E. histolytica from the non-pathogenic amebae (E. hartmanni, E. coli, Endolimax nana). ENTAMOEBA HISTOLYTICA Diagnosis  Charcot-Leyden crystals can also be seen in the stool. Charcot-Leyden crystal observed in stool specimen of a patient suffering from amebiasis (Courtesy of the Department of Parasitology, UP-CPH) ENTAMOEBA HISTOLYTICA Diagnosis  Concentration methods such as Formalin Ether/Ethyl Acetate Concentration Test (FECT) and Merthiolate Iodine Formalin Concentration Test (MIFC) are more sensitive than the DFS for detection of cysts.  The following morphologic structures are noted:  Size of the cyst  Number of nuclei  Location and appearance of the karyosome  Characteristic appearance of chromatoid bodies  Presence of cytoplasmic structures such as glycogen vacuole. ENTAMOEBA HISTOLYTICA Diagnosis  Stool culture using Robinson’s and Inoki medium is more sensitive than stool microscopy, but is not routinely available. ENTAMOEBA HISTOLYTICA Diagnosis  Differentiation between E. histolytica and E. dispar is not possible by microscopy.  This can only be done by PCR, enzyme-linked immunosorbent assay (ELISA), and isoenzyme analysis. ENTAMOEBA HISTOLYTICA Diagnosis  Detection of antibodies in the serum is still the key in the diagnosis of ALA.  In ALA, microscopic detection cannot be done because aspiration is an invasive procedure, and trophozoites are missed because they are located in the periphery of the abscess. ENTAMOEBA HISTOLYTICA Diagnosis  Serological tests for amebic disease include:  Indirect hemagglutination (IHAT)  Counter immunoelectrophoresis (CIE)  Agar gel diffusion (AGD)  Indirect fluorescent antibody test (IFAT)  ELISA ENTAMOEBA HISTOLYTICA Diagnosis  IHAT can detect antibodies of a past infection even as long as 10 years ago.  Antibodies detected by ELISA, AGD, and CIE are of short duration, lasting for a few months.  Antibodies have been demonstrated in asymptomatic intestinal infections so that serology can be used in the monitoring of a cyst carrier. ENTAMOEBA HISTOLYTICA Diagnosis  Ultrasound, computerized tomography (CT scan), and magnetic resonance imaging (MRI) are non-invasive and sensitive methods in early detection of ALA.  Ultrasound typically shows a round or oval hypoechoic area with wall echoes.  In 80% of cases, this finding is seen in the right lobe of the liver. ENTAMOEBA HISTOLYTICA Diagnosis Ultrasound showing a solitary hypoechoic mass at the right lobe of the liver suggesting ALA ENTAMOEBA HISTOLYTICA Treatment and Prognosis  Treatment of amebiasis has two objectives:  To cure invasive disease at both intestinal and extraintestinal sites  To eliminate the passage of cysts from the intestinal lumen. ENTAMOEBA HISTOLYTICA Treatment and Prognosis  Metronidazole is the drug of choice for the treatment of invasive amebiasis.  Tinidazole and secnidazole are also effective.  Diloxanide furoate is the drug of choice for asymptomatic cyst passers.  It is also given after a course of metronidazole for invasive amebiasis. ENTAMOEBA HISTOLYTICA Treatment and Prognosis  Percutaneous drainage of liver abscess  Indicated for patients who do not respond to metronidazole and who need prompt symptomatic relief of severe pain.  It is also done for those who have left lobe abscess that may rupture into the pericardium, large abscesses in danger of rupture, and multiple abscesses with a probable associated pyogenic etiology. ENTAMOEBA HISTOLYTICA Epidemiology  True prevalence of amebiasis is approximately 1 to 5% worldwide.  40,000 to 100,000 deaths recorded due to amebiasis in the world per year.  Amebiasis is the third most important parasitic disease, after malaria and schistosomiasis, and second to malaria as the top cause of mortality among parasitic protozoans. ENTAMOEBA HISTOLYTICA Epidemiology  Humans are the major reservoirs of infection with E. histolytica.  Ingestion of food and drink contaminated with E. histolytica cysts from human feces, and direct fecal-oral contact are the most common means of infection.  Amebic infection is prevalent in the Indian subcontinent, Africa, East Asia, and South and Central America. ENTAMOEBA HISTOLYTICA Epidemiology  In developing countries, prevalence depends on the level of sanitation, crowding, socio- economic status, cultural habits, and age.  In developed countries, infection is usually caused by E. dispar, and is prevalent in certain groups: immigrants, travelers from endemic countries, homosexual males (men having sex with men), HIV patients, and institutionalized people. ENTAMOEBA HISTOLYTICA Prevention and Control  Depends on integrated and community-based efforts to improve environmental sanitation, and to provide for sanitary disposal of human feces, safe drinking water, and safe food.  The proper use of latrines and practice of proper hygiene, such as washing of hands, should be emphasized.  Non potable drinking water should be boiled or filtered.  Vegetables and fruits which are eaten raw should be thoroughly washed.  Food handlers should be screened for cyst carriage, and asymptomatic cyst carriers should be treated. ENTAMOEBA HISTOLYTICA Prevention and Control  Vaccines can be a cost-effective and potent strategy for amebiasis prevention and eradication. COMMENSAL AMEBAE  Commensal amebae must be differentiated from pathogenic E. histolytica to avoid unnecessary treatment of patients infected with non-pathogenic species.  The presence of commensal amebae in the stools of an individual is significant for two reasons:  The amebae may be mistaken for the pathogenic Entamoeba histolytica  It is an indication of fecal contamination of food or water. COMMENSAL AMEBAE Parasite Biology  The three genera of commensal intestinal amebae can be differentiated through the morphological features of their nuclei  The genus Entamoeba has a spherical nucleus with a distinct nuclear membrane lined with chromatin granules and a small karyosome found near the center of the nucleus.  The genus Endolimax has a vesicular nucleus with a relatively large, irregularly- shaped karyosome anchored to the nucleus by achromatic fibrils. COMMENSAL AMEBAE Parasite Biology  The genus Iodamoeba is characterized by a large, chromatin-rich karyosome surrounded by a layer of achromatic globules and anchored to the nuclear membrane by achromatic fibrils. COMMENSAL AMEBAE Parasite Biology  All species have the following stages: trophozoite, precyst, cyst, and metacystic trophozoite (except Entamoeba gingivalis which has no cyst stage, and does not inhabit the intestines)  Humans are infected by commensal intestinal amebae through ingestion of viable cysts in food or water.  Cysts pass through the acidic stomach and remain viable because of protective cyst walls. COMMENSAL AMEBAE Parasite Biology  Excystation occurs in the alkaline environment of the lower small intestines.  Metacystic trophozoites colonize the large intestines and live on the mucus coat covering the intestinal mucosa  They are non-invasive and do not cause disease.  Reproduction is by binary fission of the trophozoites.  Encystation occurs in lower colon COMMENSAL AMEBAE ENTAMOEBA DISPAR Entamoeba dispar is morphologically similar to E. histolytica, but their DNA and ribosomal RNA are different. ENTAMOEBA MOSHKOVSKII  First detected in sewage  Reported in some areas, such as North America, Italy, South Africa, Bangladesh, India, Iran, and Australia.  It is a non-pathogenic species that is morphologically indistinguishable from E. histolytica and E. dispar, but differs from them biochemically and genetically.  Physiologically unique being osmotolerant, able to grow at room temperature (25-30°C optimum), able to survive at temperatures ranging from 0 to 41°C. ENTAMOEBA HARTMANNI  The appearance of E. hartmanni is relatively similar to that of E. histolytica apart from its smaller size.  Trophozoites measure from 3 to 12 μm in diameter (compared to E. histolytica measuring 12-60 μm).  Mature cysts measure 4 to 10 μm, are quadrinucleated like E. histolytica, and have rod-shaped chromatoid material with rounded or squared ends.  Unlike E. histolytica, E. hartmanni does not ingest red blood cells. ENTAMOEBA COLI  Entamoeba coli is cosmopolitan in distribution, and is considerably more common than other human amebae.  Trophozoites of E. coli measure 15 to 50 μm in diameter. ENTAMOEBA COLI Trophozoite can be differentiated from E. histolytica by the following features:  A more vacuolated or granular endoplasm with bacteria and debris, but no red blood cells  A narrower, less-differentiated ectoplasm  Broader and blunter pseudopodia used more for feeding than locomotion  More sluggish, undirected movements  Thicker, irregular peripheral chromatin with a large, eccentric karyosome in the nucleus ENTAMOEBA COLI Entamoeba coli trophozoite (Courtesy of Department of Parasitology, UP-CPH) ENTAMOEBA COLI  Cyst can be differentiated from E. histolytica by:  Its larger size (10 to 35 μm in diameter)  More nuclei (eight versus four in E. histolytica)  More granular cytoplasm, splinter-like chromatoidal bodies  Iodine staining reveals dark-staining, perinuclear masses, which are actually glycogen.  Its location, surrounding the nucleus, is more characteristic of E. coli compared to E. histolytica. ENTAMOEBA POLECKI  Parasite found in the intestines of pigs and monkeys.  Rarely, it can infect humans, though a high prevalence (19%) was reported in some parts of Papua New Guinea  In these areas, both pig-to-human and human-to-human transmission may exist. ENTAMOEBA POLECKI  Like E. coli, motility of trophozoites of E. polecki is sluggish.  A small karyosome is centrally located in the nucleus.  Cyst can be distinguished from E. histolytica by its uninucleated, and the presence of chromatoidal bars that are frequently angular or pointed.  In stained fecal smears, the nuclear membrane and karyosome are very prominent. ENTAMOEBA CHATTONI  Found in apes and monkeys  Morphologically identical to E. polecki.  It has been detected in eight human infections.  Identification of E. chattoni was done via isoenzyme analysis. ENTAMOEBA GINGIVALIS  Can be found in the mouth.  Lives on the surface of gum and teeth, in gum pockets, and sometimes in the tonsillar crypts.  They are abundant in cases of oral disease.  The trophozoite measures 10 to 20 μm.  It moves quickly, and has numerous blunt pseudopodia.  Food vacuoles that contain cellular debris (mostly leukocytes, which is characteristic of this species) and bacteria are numerous. ENTAMOEBA GINGIVALIS  This species has no cyst stage  Transmission is most probably direct: through kissing, droplet spray, or by sharing utensils. ENDOLIMAX NANA  Trophozoites are small, with a diameter of 5 to 12 μm, and exhibit sluggish movement.  They have blunt, hyaline pseudopodia  The nucleus has a large, irregular karyosome  Food vacuoles found in the cytoplasm may contain bacteria.  Cysts measure about the same size as trophozoites, and are quadrinucleated when mature. IODAMOEBA BÜTSCHLII  The trophozoite averages 9 to 14 μm in diameter (ranging from 4-20 μm)  It is identified by its characteristic large, vesicular nucleus with a large, central karyosome, surrounded by achromatic granules  There are no peripheral chromatin granules on the nuclear membrane  The cyst is about 9 to 10 μm in diameter (ranging from 6-16 μm)  Uninucleated  Has a large glycogen body which stains dark brown with iodine IODAMOEBA BÜTSCHLII Iodamoeba bütschlii cyst (Courtesy of the Department of Parasitology, UP-CPH) DIAGNOSIS  Diagnosis is done through stool examination.  Formalin ether/ethyl acetate concentration technique (FECT) and iodine stain are useful to differentiate the species.  For E. gingivalis, a swab between the gums and teeth is examined for trophozoites.  Cysts are recovered from formed stools, while trophozoites are recovered from watery or semi-formed stools.  Trophozoites are best demonstrated by direct fecal smear.  Cysts are best demonstrated by the use of concentration techniques like FECT and zinc sulfate flotation TREATMENT  No treatment is necessary because these amebae do not cause disease. PREVENTION AND CONTROL  Contraction of the organism may be prevented through proper disposal of human waste and good personal hygiene. FREE-LIVING PATHOGENIC AMEBAE  Acanthamoeba spp.  Naegleria spp. ACANTHAMOEBA SPP. Parasite Biology  Ubiquitous, free-living ameba  Etiologic agent of Acanthamoeba keratitis (AK) and granulomatous amebic encephalitis (GAE)  Characterized by an active trophozoite stage with characteristic prominent “thorn-like” appendages (acanthopodia); and a highly resilient cyst stage into which it transforms when environmental conditions are not favorable. ACANTHAMOEBA SPP. Parasite Biology  It is an aquatic organism that is found in a myriad of natural and artificial environments, and can survive even in contact lens cleaning solutions.  Motile trophozoites feed on gram-negative bacteria, blue-green algae, or yeasts  Can adapt to feed on corneal epithelial cells and neurologic tissue through phagocytosis and secretion of lytic enzymes.  Reproduce by binary fission ACANTHAMOEBA SPP. Parasite Biology  Trophozoites exhibit a characteristic single large nucleus with a centrally- located, densely staining nucleolus; a large endosome; finely granulated cytoplasm; and a large contractile vacuole.  Small, spiny filaments for locomotion known as acanthapodia are evident on phase- contrast microscopy ACANTHAMOEBA SPP. Parasite Biology  Has only two stages, cysts and trophozoites, in its life cycle.  No flagellated stage exists as part of the life cycle.  The trophozoites replicate by mitosis (nuclear membrane does not remain intact).  The trophozoites are the infective stage, although both cysts and trophozoites gain entry into the body through various means.  Entry can occur through the eye, the nasal passages to the lower respiratory tract, or ulcerated or broken skin ACANTHAMOEBA SPP. Parasite Biology  Recently, Acanthamoeba spp. have been implicated as possible reservoir hosts for medically important bacteria such as Legionella spp., mycobacteria, and gram- negative bacilli such as E. coli. ACANTHAMOEBA SPP. Acanthamoeba trophozoite exhibiting characteristic acanthopodia (Accessed from www.dpd.cdc.gov/dpdx) ACANTHAMOEBA SPP. Pathogenesis and Clinical Manifestations Acanthamoeba Keratitis  Acanthamoeba was first described as an opportunistic ocular surface pathogen causing keratitis in 1974.  AK is associated with the use of improperly disinfected soft contact lenses, particularly those which are rinsed with tap water or contaminated lens solution.  An immunocompromised state contributes to increased susceptibility to infection, and may lead to disseminated disease in the lungs and brain (GAE). ACANTHAMOEBA SPP. Pathogenesis and Clinical Manifestations Acanthamoeba Keratitis  Symptoms include severe ocular pain and blurring of vision.  Corneal ulceration with progressive corneal infiltration may occur.  Primary amebic infection or secondary bacterial infection may lead to hypopyon formation.  Progression of infection may cause scleritis and iritis, and may ultimately lead to vision loss. ACANTHAMOEBA SPP. Pathogenesis and Clinical Manifestations Granulomatous Amebic Encephalitis  Acanthamoeba was documented as the causative agent of human GAE by Stamm in 1972.  GAE usually occurs in immunocompromised hosts including the chronically ill and debilitated, and those on immunosuppressive agents such as chemotherapy and anti- rejection medications.  AIDS epidemic in the 1980’s dramatically increased the numbers of person with GAE, but these numbers have since fallen with the advent of highly effective antiretroviral therapy. ACANTHAMOEBA SPP. Pathogenesis and Clinical Manifestations Granulomatous Amebic Encephalitis  Signs and symptoms are generally related to destruction of brain tissue and the associated meningeal irritation.  Systemic manifestations early in the course include fever, malaise, and anorexia.  Neurologic symptoms may include increased sleeping time, severe headache, mental status changes, epilepsy, and coma.  Neurologic findings depending on the location of the lesions include hemiparesis, blurring of vision, diplopia, cranial nerve deficits, ataxia, and increased intracranial pressure. ACANTHAMOEBA SPP. Pathogenesis and Clinical Manifestations Granulomatous Amebic Encephalitis  Entry of Acanthamoeba into the central nervous system is still incompletely understood.  From a primary site of infection in the skin or lungs, the likely route of invasion is hematogenous.  Direct infection through the olfactory valves has also been proposed, but not conclusively demonstrated. ACANTHAMOEBA SPP. Pathogenesis and Clinical Manifestations Granulomatous Amebic Encephalitis  Gross examination of neural tissue postmortem reveals cerebral hemispheres that are edematous and soft, with areas of hemorrhage and focal abscesses.  The most affected areas of the brain are the posterior fossa structures, thalamus, and the brainstem.  In the affected areas, the leptomeninges are opaque and exhibit purulent exudates and vascular congestion. ACANTHAMOEBA SPP. Pathogenesis and Clinical Manifestations Granulomatous Amebic Encephalitis  The incubation period from initial inoculation is approximately 10 days  Subacute and chronic clinical course of infection that lasts for several weeks to several months.  The clinical manifestations of disease include decreased sensorium, altered mental status, meningitis, and neurologic deficits.  The natural course of the disease eventually results in coma and death (within 3 to 40 days) ACANTHAMOEBA SPP. Diagnosis Acanthamoeba keratitis  Diagnosed by epithelial biopsy or corneal scrapings for recoverable ameba with characteristic staining patterns on histologic analysis.  Amebae have also been isolated from the contact lens and lens solution of patients. ACANTHAMOEBA SPP. Diagnosis  Diagnosis of GAE is usually made postmortem in most cases.  Recovery of ameba from cerebrospinal fluid is exceedingly rare, and imaging results are generally nonspecific.  Specific diagnosis depends on demonstrating the trophozoites or cysts in tissues using histopathologic stains and microscopy. ACANTHAMOEBA SPP. Treatment Acanthamoeba keratitis  Only surgical excision of the infected cornea with subsequent corneal transplantation was curative  Deep lamellar keratectomy is the procedure of choice.  Early recognition of AK coupled with aggressive combination anti-amebic agents can preclude the need for extensive surgery.  Clotrimazole combined with pentamidine, isethionate, and neosporin  Polyhexamethylene biguanide, propamidine, dibromopropamidine isethionate, neomycin, paromomycin, polymyxin B, ketoconazole, miconazole, and itraconazole. ACANTHAMOEBA SPP. Treatment Acanthamoeba keratitis  Topical corticosteroids should be avoided, as this retards the immune response.  Advanced AK usually requires debridement ACANTHAMOEBA SPP. Treatment Granulomatous Amebic Encephalitis  A few patients have shown good responses to combinations of amphotericin B, pentamidine isethionate, sulfadiazine, flucytosine, fluconazole or itraconazole.  One liver transplant patient survived after decompressive frontal lobectomy and treatment with amphotericin, cotrimoxazole, and rifampin. ACANTHAMOEBA SPP. Epidemiology  Acanthamoeba spp. have been isolated from a multitude of natural and artificial aquatic environments including fresh and salt water, sewage, hospital equipment, and contact lenses and lens solution.  The first case of AK was recognized in the Philippines in the 1990s from a patient from the Philippine General Hospital, and samples obtained from the patient was shown to cause GAE in mice. ACANTHAMOEBA SPP. Prevention and Control  The ubiquitious nature of Acanthamoeba spp. makes exposure unavoidable.  A robust immune system is able to prevent infection, except in relatively immunocompromised sites such as the cornea.  Meticulous contact lens hygiene is essential in avoiding infection, and rinsing contact lenses in tap water should be avoided.  Prolonged heating and boiling kill amebic trophozoites and cyst forms.  Immunocompromised persons should be aware of the risk of infection  NAEGLERIA SPP. Parasite Biology  Naegleria spp. are free-living protozoans with two vegetative forms (trophozoites):  Ameba (trophozoite form)  Flagellate (swimming form)  NAEGLERIA SPP. Parasite Biology  Ameboid trophozoite is the only trophozoite found in humans  The ameboid trophozoites measure 10 to 35 μm but when rounded are usually 10 to 15 μm in diameter.  The cytoplasm is granular and contains many vacuoles  The single nucleus is large and has a large, dense karyosome and lacks peripheral chromatin  NAEGLERIA SPP. Parasite Biology  A dormant cyst form is produced when conditions are not favorable.  Transformation from the trophozoite to the flagellate form may facilitate more rapid movement toward food sources.  NAEGLERIA SPP. Parasite Biology  Naegleria spp. are thermophilic organisms which thrive best in hot springs and other warm aquatic environments.  Able to survive in elevated temperatures and reproduces rapidly in temperatures above 30°C.  Both nonpathogenic and pathogenic forms exist.  Only Naegleria fowleri has been reported to consistently cause disease in humans  NAEGLERIA SPP. Parasite Biology Naegleria fowleri trophozoites in ameboid (left) and ameboflagellate (right) forms (Accessed from www.dpd.cdc.gov/dpdx)  NAEGLERIA SPP. Parasite Biology  Naegleria fowleri has three stages, cysts, trophozoites, and flagellated forms, in its life cycle.  The trophozoites replicate by promitosis (nuclear membrane remains intact) and can turn into temporary non-feeding flagellated forms, which usually revert back to the trophozoite stage.  Trophozoites infect humans or animals by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves.  NAEGLERIA SPP. Parasite Biology  N. fowleri trophozoites are found in cerebrospinal fluid (CSF) and tissue, while flagellated forms are occasionally found in CSF.  Cysts are not seen in brain tissue NAEGLERIA SPP. NAEGLERIA SPP. Pathogenesis and Clinical Manifestations  Primary amebic meningoencephalitis (PAM)  Rapidly destructive and fatal meningoencephalitis  In contrast to GAE which is predominantly an opportunistic infection, PAM usually occurs in previously healthy adults with a history of swimming.  Therefore, in contrast to Acanthamoeba which is largely an opportunistic organism, N. fowleri is considered a true pathogen NAEGLERIA SPP. Pathogenesis and Clinical Manifestations  Primary amebic meningoencephalitis (PAM)  Most cases of PAM have occurred in young, healthy persons who swim in contaminated water.  The route of entry is through invasion of organisms through the olfactory bulb after accidental inhalation of water containing the organisms.  The sustentacular cells of the olfactory neuroepithelium are thought to phagocytose the amebae and transport these through the cribriform plate and into the brain. NAEGLERIA SPP. Pathogenesis and Clinical Manifestations  Primary amebic meningoencephalitis (PAM)  Mechanisms of disease causation include secretion of lytic enzymes, membrane pore-forming proteins, factors which induce apoptosis, and direct feeding on cells by the amebae. NAEGLERIA SPP. Pathogenesis and Clinical Manifestations  Primary amebic meningoencephalitis (PAM)  PAM presents as fever, nausea, vomiting, headache, nuchal rigidity, and mental status changes, with rapid progression to coma and death.  Characteristic cerebrospinal fluid findings include elevated white blood cell count with neutrophilic predominance, high protein, and low glucose. NAEGLERIA SPP. Pathogenesis and Clinical Manifestations  Primary amebic meningoencephalitis (PAM)  Post-mortem examination of infected brain shows hemorrhagic necrosis, particularly of the olfactory bulbs, congestion and edema of neural tissue.  Leptomeninges are inflamed and congested  Microscopic examination shows a fibrinopurulent exudate consisting mostly of neutrophils in the leptomeninges and brain tissue, and pockets of amebae with scant inflammatory exudates in necrotic areas. NAEGLERIA SPP. Pathogenesis and Clinical Manifestations  Primary amebic meningoencephalitis (PAM)  Death usually occurs as a result of cerebral or cerebellar herniation as a result of increased intracranial pressure. NAEGLERIA SPP. Diagnosis  Suspected in persons with a compatible history of exposure and a rapidly progressive meningoencephalitis.  In the past, definitive diagnosis of PAM was based on demonstration of characteristic trophozoites in the brain and cerebrospinal fluid. NAEGLERIA SPP. Diagnosis  Naegleria trophozoites can be identified by the presence of blunt, lobose pseudopodia and directional motility.  PCR and immunostaining  Serology utilizing ELISA is less useful in diagnosing active infection since healthy individuals especially in endemic areas have been shown to have positive antibody titers. NAEGLERIA SPP. Treatment  Amphotericin B in combination with clotrimazole is synergistic, and has been successfully used to treat PAM.  Amphotericin B produces deleterious changes in the nucleus and mitochondria of the ameba, decreases the number of food vacuoles, and increases the formation of autophagic vacuoles. NAEGLERIA SPP. Treatment  Ameba exposed to amphotericin B exhibit decreased pseudopod formation and form blebs on the plasma membrane.  Newer agents such as azithromycin and voriconazole have been shown to be active against N. fowleri, both in vitro and in vivo. NAEGLERIA SPP. Epidemiology  Studies on local Naegleria have identified a new species which is morphologically indistinguishable but biochemically distinct from other known species.  Isolates from a thermally-polluted stream, an artificially-heated swimming pool, and from the brain aspirate of a young patient have all yielded a single species, N. philippinensis.  Only one case of PAM has been reported locally, in a young male with a history of swimming in fresh water.  Responded well to amphotericin B infusion. NAEGLERIA SPP. Prevention and Control  Avoiding immersion of the head and accidental inhalation of water should be practiced in endemic areas and in hot springs.  No known cases of PAM have resulted from drinking ameba-infected water. NAEGLERIA SPP. Prevention and Control  Naegleria fowleri is easily killed by chlorination of water at 1 ppm or higher.  Infection has been reported from swimming in contaminated water with inadequate chlorination, and so recommendations for appropriate decontamination of swimming water should be followed, especially in areas of high prevalence.