Other Protozoa

Chapter 50


Other Protozoa






Free-Living Amebae


Infections caused by small, free-living amebae belonging to the genera Naegleria, Acanthamoeba, and Balamuthia are generally not very well-known or recognized clinically. Also, methods for laboratory diagnosis are unfamiliar and not routinely offered by most laboratories. However, approximately 310 cases of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri and more than 150 cases of granulomatous amebic encephalitis (GAE) caused by Acanthamoeba spp. and Balamuthia mandrillaris (including several cases in patients with acquired immunodeficiency syndrome [AIDS]) have been documented. Other infections caused by these organisms result in Acanthamoeba keratitis, now numbering more than 750 cases and related primarily to poor lens care in contact lens wearers. Additionally, both Acanthamoeba spp. and B. mandrillaris can cause cutaneous infections in humans. Sappinia pedata, a free-living ameba normally found in soil contaminated with the feces of elk and buffalo, was identified in an excised brain lesion from a 38-year-old immunocompetent man who developed a frontal headache, blurry vision, and loss of consciousness following a sinus infection. Additionally, Paravahlkampfia francinae, a new species of the free-living ameba genus Paravahlkampfia, was recently isolated from the cerebrospinal fluid (CSF) of a patient with a headache, sore throat, and vomiting, symptoms typical of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri from the environment.



Naegleria Fowleri


General Characteristics


There are both trophozoite and cyst stages in the life cycle, with the stage present primarily dependent on environmental conditions. Trophozoites can be found in water or moist soil and can be maintained in tissue culture or other artificial media. The amebae may enter the nasal cavity by inhalation or aspiration of water, dust, or aerosols containing the trophozoites or cysts. N. fowleri is incapable of survival in clean, chlorinated water. Following inhalation or aspiration, the organisms then penetrate the nasal mucosa, probably through phagocytosis of the olfactory epithelium cells, and migrate via the olfactory nerves to the brain.


The trophozoites can occur in two forms: ameboid and flagellate (Table 50-1, Figure 50-1). The size ranges from 7 to 35 µm. The diameter of the rounded forms is usually 15 µm. There is a large, central karyosome and no peripheral nuclear chromatin. The cytoplasm is somewhat granular and contains vacuoles. The ameboid form organisms change to the transient, pear-shaped flagellate form when they are transferred from culture or teased from tissue into water and maintained at a temperature of 27° to 37° C. These flagellate forms do not divide, but when the flagella are lost, the ameboid forms resume reproduction. Cysts are generally round, measuring from 7 to 15 µm with a thick double wall.



TABLE 50-1


Free-Living Amebae Causing Disease in Humans
































































  Acanthamoeba Balamuthia Naegleria
Morphologya
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Trophozoite

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Cyst

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Keratitis

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Acanthamoeba in brain

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Trophozoite

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Cyst

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Balamuthia in brain

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Trophozoite

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Flagellated form
Disease parameter Granulomatous amebic encephalitis (GAE) Acanthamoeba keratitis Cutaneous lesions
Sinusitis
GAE Primary amebic meningoencephalitis
(PAM)
General disease description Chronic, protracted, slowly progressive CNS infection (may involve lungs); generally associated with individuals with underlying diseases Painful, progressive, sight-threatening corneal disease; patients generally immunocompetent Most common in patients with AIDS, with or without CNS involvement; those receiving immunosuppressive therapy for organ transplantation Chronic, protracted, slowly progressive CNS infection (may involve lungs); generally associated with individuals with underlying diseases Rare, but nearly always fatal infection; migration of amebae to brain through olfactory nerve; symptoms can mimic bacterial meningitis; death usually occurs 3-7 days after onset of symptoms; clinical suspicion based on history critical
Entry into body Olfactory epithelium, respiratory tract, skin, sinuses Corneal abrasion Skin, sinuses, respiratory tract Olfactory epithelium, skin, respiratory tract Olfactory epithelium
Incubation period Weeks to months Days Weeks to months Weeks to months Days
Clinical symptoms Confusion, headache, stiff neck, irritability Blurred vision, photophobia, inflammation, corneal ring, pain Skin lesions, nodules, sinus lesions, sinusitis Slurred speech, muscle weakness, headache, nausea, seizures Headache, nausea, vomiting, confusion, fever, stiff neck, seizures, coma
Disease pathology Focal necrosis, granulomas Corneal ulceration Granulomatous reaction in skin, inflammation Multiple necrotic foci, inflammation, cerebral edema Hemorrhagic necrosis
Diagnostic methods Brain biopsy, CSF smear/wet prep, culture, indirect immunofluorescence on tissue,b PCRb Corneal scrapings or biopsy, stain with calcofluor white, culture, confocal microscopy Skin lesion biopsy, culture, indirect immunofluorescence of tissuea Brain biopsy, culture on mammalian cells, indirect immunofluorescence of tissuea Brain biopsy, CSF wet prep, culture, indirect immunofluorescence of tissue,a PCRa


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aAcanthamoeba in brain and Balamuthia in brain courtesy Dr. Govinda Visvesvara, Centers for Disease Control and Prevention.


bIndirect immunofluorescence on tissue and PCR methods available from Centers for Disease Control and Prevention.




Pathogenesis and Spectrum of Disease


Primary amebic meningoencephalitis (PAM) caused by N. fowleri is an acute, suppurative infection of the brain and meninges (Figure 50-2). With extremely rare exceptions, the disease is rapidly fatal in humans. The period between organism contact and onset of symptoms such as fever, headache, and rhinitis varies from a few days to 2 weeks. Early symptoms include vague upper respiratory tract distress, headache, lethargy, and occasionally olfactory problems. The acute phase includes sore throat; a stuffy, blocked, or discharging nose; and severe headache. Progressive symptoms include pyrexia, vomiting, and stiffness of the neck. Mental confusion and coma usually occur approximately 3 to 5 days before death, which is usually caused by cardiorespiratory arrest and pulmonary edema.



PAM resembles acute bacterial meningitis, and these conditions may be difficult to differentiate. Unfortunately, if the CSF Gram stain is interpreted incorrectly as a false positive, the resulting antibacterial therapy has no impact on the amebae and the patient will usually die within a few days.



Laboratory Diagnosis


Routine Methods


Clinical and laboratory data usually cannot be used to differentiate pyogenic meningitis from PAM. A high index of suspicion is often critical for early diagnosis. Most cases are associated with exposure to contaminated water through swimming or bathing. There is normally an incubation period of 1 day to 2 weeks, and then a course of 3 to 6 days, most often ending in death.


Analysis of the CSF will show decreased glucose and increased protein concentrations. The leukocyte count will range from several hundred to >20,000 cells per mm3. Although Gram stains and bacterial cultures of CSF will be negative, serious patient complications can occur as the result of incorrect therapy if false-positive Gram stains are reported.


A confirmed diagnosis is made by the identification of amebae in the CSF or in biopsy specimens. CSF should be placed on a slide, under a cover slip, and observed for motile trophozoites; smears can be stained with any of the blood stains. It is important not to mistake leukocytes for actual organisms or vice versa. This type of misidentification often occurs when using a counting chamber and the amebae sink to the bottom and round up, hence the recommendation to use just a regular slide and cover slip. Depending on the temperature and lag time between specimen collection and examination, motility may vary. Slides may be warmed slightly to improve motility. The most important differential characteristic is the spherical nucleus with a large karyosome.


Specimens should never be refrigerated before examination, and CSF should be centrifuged at a slow speed (250× g). If N. fowleri is the causative agent, only trophozoites are normally seen, whereas cysts and trophozoites can be seen with Acanthamoeba spp.





Acanthamoeba Spp.


General Characteristics


Unlike N. fowleri, Acanthamoeba spp. do not have a flagellate stage in the life cycle, only the trophozoite and cyst. Several species of Acanthamoeba cause granulomatous amebic encephalitis (GAE), primarily in immunosuppressed, chronically ill, or otherwise debilitated individuals. These patients usually have no relevant history involving freshwater exposure. Acanthamoeba spp. also cause amebic keratitis, and it is estimated that the incidence in the United States may be one to two cases per million contact lens users. Apparently, the incidence of Acanthamoeba keratitis in the United Kingdom is 15 times higher than that in the United States and 7 times higher than that in Holland.


Motile organisms have spine-like pseudopods; there is a wide organism size range (25 to 40 µm), with the average diameter of the trophozoites being 30 µm. The nucleus has the typical large karyosome, similar to that found in N. fowleri. This morphologic characteristic can be seen on a wet preparation.


The cysts are usually round with a single nucleus, also having the large karyosome as in the trophozoite nucleus. The double wall is usually visible, with the slightly wrinkled outer cyst wall and what has been described as a polyhedral inner cyst wall. This cyst morphology is identifiable in organisms cultured on agar plates.



Pathogenesis and Spectrum of Disease


GAE


Meningoencephalitis caused by Acanthamoeba spp. may present as an acute suppurative inflammation of the brain and meninges similar to N. fowleri infection. The incubation period of GAE is unknown; several weeks or months are probably necessary to establish disease. The clinical course tends to be subacute or chronic and is usually associated with trauma or underlying disease, not as a result of swimming. GAE may present with symptoms of confusion, dizziness, drowsiness, nausea, vomiting, headache, lethargy, stiff neck, seizures, and sometimes hemiparesis. Unlike PAM caused by N. fowleri, both trophozoites and cysts are found throughout the tissue. Also, dissemination to other tissues such as the liver, kidneys, trachea, and adrenals can occur in immunocompromised individuals; or additional unusual sites also include the ear and necrotic bone from a bone graft of the mandible. Some patients, especially those with AIDS, can develop erythematous nodules, chronic ulcerative skin lesions, or abscesses.



Keratitis


Acanthamoeba spp. also cause keratitis and corneal ulceration. Clinicians need to consider acanthamoebic infection in the differential diagnosis of eye infections that are not responding to bacterial, fungal, or viral therapy. These infections are often due to direct exposure of the eyes to contaminated materials or solutions. Use of contact lenses is the leading risk factor for keratitis. Conditions that are linked with disease include the use of home-made saline solutions, poor contact lens hygiene, and corneal abrasions. A contact lens can act as a mechanical vector for transport of amebae present in the storage case onto the cornea. Subsequent multiplication and invasion of the tissue may occur. Decreased corneal sensation has contributed to the misdiagnosis of Acanthamoeba keratitis as herpes simplex keratitis. Acanthamoeba keratitis may be present as a secondary or opportunistic infection in patients with herpes simplex keratitis. Unfortunately, as a result, treatment can be delayed for 2 weeks to 3 months.



Laboratory Diagnosis


Routine Methods


The most effective culture approach uses non-nutrient agar plates with Page’s saline and an overlay growth of Escherichia coli on which the amebae feed. Tissue stains are also effective, and cysts isolated from cultures can be stained with Gomori’s silver methenamine, periodic acid-Schiff, and calcofluor white. Identification of Acanthamoebae in ocular samples and other tissues can be difficult, even for trained laboratory professionals; in histologic preparations, the organisms appear similar to keratoplasts, as well as neutrophils and monocytes. It has been estimated that up to 70% of clinical Acanthamoeba keratitis cases are misdiagnosed as viral keratitis. Also, the average time to diagnosis of keratitis attributable to Acanthamoeba infection can average 2.5 weeks longer for non–contact lens wearers than for contact lens users.



Other Methods


CSF or bronchoalveolar lavage fluid cytospin preparations can be used to look for amebae in patients with GAE or respiratory symptoms. The characteristic morphology of the Acanthamoeba trophozoites, such as the prominent nucleolus, contractile vacuole, and cytoplasmic vacuoles, can be seen more easily using trichrome or hematoxylin and eosin stains on fixed preparations after cytocentrifugation.


In the differential diagnosis of GAE, other space-occupying lesions of the central nervous system (CNS) (e.g., tumor, abscess, fungal infection) must also be considered. Predisposing conditions include Hodgkin’s disease, diabetes, alcoholism, pregnancy, and corticosteroid therapy. Organisms have also been found in the adrenal gland, brain, eyes, kidneys, liver, pancreas, skin, spleen, thyroid gland, and uterus.


In infections caused by Acanthamoeba spp., periodic acid-Schiff stains the cyst wall red and methenamine silver stains the cyst black. Normally, Naegleria and Acanthamoeba isolates are identified to the species level by a reference laboratory, such as the Centers for Disease Control and Prevention, using indirect fluorescent antibody procedures with a monoclonal or polyclonal antibody.

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Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on Other Protozoa

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