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Emerging trends in free-living amebic infections of the brain: implications for organ transplantation.

Free-living amebae of the genera Acanthamoeba, Balamuthia, Naegleria, and Sappinia are rare causes of infectious diseases in humans, with the exception of Acanthamoeba keratitis (AK), which is reported in more than one to two cases per 1 million contact lens wearers in the US annually. (1,2) Unlike several Acanthamoeba species, only one species of Naegleria, N. fowleri, is known to infect humans by causing an acute, fulminant, usually lethal, brain infection known as primary amebic meningoencephalitis (PAM). (1) Sappinia pedata, a recently identified free-living ameba that lives in soil and domestic animal feces, has caused a single, non-fatal case of non-granulomatous amebic encephalitis in an immunocompetent Texas farmer. (1)

Balamuthia mandrillaris is another opportunistic, free-living ameba that, like Acanthamoeba, can cause chronic skin lesions and granulomatous amebic encephalitis (GAE) in individuals with either compromised or competent immune systems. (2) Both Acanthamoeba and Balamuthia-caused GAE are often characterized by nodular or ulcerating granulomatous skin lesions in soil-contaminated wounds or in similar lesions that may appear spontaneously following organ transplantation. (1,2) The objectives of this review and descriptive epidemiological analysis were to describe the epidemiology, diagnosis, and management of the three most common free-living amebic infections of the brain which are compared by their life cycles in Figure 1.


The only cases of free-living amebic meningoencephalitis included in the analytical case reviews were cases with CDC laboratory-confirmed detection of N. fowleri or B. mandrillaris life cycle forms by immunohistopathological techniques, isolation by culture, or by species-specific DNA detection by polymerase chain reaction (PCR) in cerebrospinal fluid (CSF), brain biopsy, or brain necropsy tissue.

Sources of US cases of PAM came from the registry of the CDC's Naegleria Workgroup, which confirmed 121 cases of PAM in the US during the period, 1937-2007. (3) Sources of US cases of Balmuthia GAE, or balamuthiasis, came from the CDC or its affiliated state departments of public health and the California Encephalitis Project (CEP), a joint project launched in 1998 by the California Department of Public Health and the CDC. Similar descriptive analyses were conducted for all CDC laboratory-confirmed cases of GAE caused by B. mandrillaris (N = 28) in the US during the period, 1994-2010. Continuous variables were tested for significant differences by unpaired, two-tailed t-tests, and proportions were tested for significant differences by the "ratio.test" using the Poisson distribution. The Poisson distribution was used to model the number of case clusters per year of PAM and the number of deaths per year from PAM. Both of these two rates were compared for the time periods, 1937-1976 vs. 1977-2007. These two time periods were selected for comparative analyses because no CDC-confirmed PAM cases were reported in 1976, a break-point year; and each of the time periods had durations of at least three decades. Hypothesis tests on these time period comparisons of PAM cases were performed using the "ratio.test" function from the Internet-available R Statistical Package. Statistical significance was indicated by p-values less than or equal to 0.05.

Results: Primary amebic meningoencephalitis (PAM)

N. fowleri, the single causative agent of PAM, is a free-living, thermophilic amoeboflagellate that thrives in many types of warm freshwater, including shallow inland lakes, geothermal springs, warm water discharges from electrical power plants, and even domestic water supplies in the US and throughout the temperate world. (1) N. fowleri feeds on bacteria and organic debris in freshwater and exists in three life forms - the environmentally stable cyst form and two trophozoite forms, ameboid and ameboflagellate (Figure 1). The ameboid trophozoites of N. fowleri measure from 10 um in diameter when rounded and to more than 40 um in diameter when ameboid. (1) They typically invade humans via intact or disrupted nasal mucosa, cross the cribriform plate, migrate along the basilar brain surfaces from the olfactory bulbs and tracts to the cerebellum, deeply penetrate the cortex to the periventricular system, and incite a purulent meningoencephalitis. Rapid cerebral edema ensues with early, typically fatal, uncal and cerebellar herniation. (1) An alternative, uncommon human invasion route for N. fowleri trophozoites may be through an infection-weakened or a traumatically ruptured tympanic membrane (TM), which affords invasive forms access to the brain via the middle and inner ear to the acoustic nerve tract and on to the basilar brainstem. (1)

In this study, a statistical analysis of the 121 PAM cases by Poisson distribution to model the number of deaths per year from PAM and the number of case clusters per year of PAM was conducted in order to confirm or refute earlier findings. The background frequency of PAM cases in the US was zero to three cases per year over the entire 60-year study period, 1937-2007; three of the six cases (50%) in the 2007 cluster investigated by the CDC were males (ages 10, 11, and 22 years) who had been wakeboarding in freshwater lakes. (1) Descriptive analysis of all CDC-documented US cases of PAM during 1937-2007 identified the following presenting clinical characteristics for PAM in the US: (1) male gender; (2) recreational freshwater exposures; (3) summer seasonal exposures; and (4) exposures in a southern-tier US state. There were no differences in the frequency of cases per year, per period, or in the high case fatality rates per year, per period (range = 98%-100%). However, there were more cases in the more recent study period, significantly more deaths in the more recent period (p < 0.001), and significantly more clusters of four or more cases in the more recent study period (p = 0.001).

Initial screening laboratory studies are nonspecific in all free-living amebic infections of the brain, and bacterial blood cultures and peripheral blood Gram stains will be negative. (1) Neuroimaging studies in PAM are also nonspecific and may be normal on initial cranial computerized tomographic (CT) and magnetic resonance imaging (MRI) scans. (4) Subsequent neuroimaging findings may include basilar leptomeningeal enhancement, massive cerebral edema, evidence of elevated intracranial pressure (midline shift, compressed ventricles, compressed brainstem and basilar cisterns, absence of subarachnoid spaces), and multifocal parenchymal lesions, often with evidence of hemorrhagic infarction or necrosis. (4)

Although usually futile, successful management strategies for PAM have included combinations of cerebral edemareducing therapies (corticosteroids, moderate hyperventilation, diuresis, hypertonic saline), specific pharmacotherapy with antifungals (amphotericin B, miconazole, voriconazole) and synergistic antibiotics (rifampin, azithromycin), and the addition of several effective investigational agents. (5,6) Some experimental therapies that have shown some promise in treating PAM include the phenothiazines; chlorpromazine and thioridazine; and miltefosine, a phosphocholine analog, more commonly used to treat visceral leishmaniasis. (6) Kim and co-investigators demonstrated that combination of amphotericin B, chlorpromazine, and miltefosine inhibited the growth of N. fowleri in vitro, and significantly lengthened survival times in mice experimentally infected with N. fowleri with increasing doses of the combination. (5) Although not FDA-approved for use in PAM, miltifosine is available to clinicians treating suspected PAM cases who contact the CDC Emergency Operations Center.

Three recently emerging trends in the epidemiology of PAM have now been reported that should raise awareness of PAM among all clinicians caring for patients with fulminant aseptic meningitis-like presentations, including (1) an expanding geographic range of PAM beyond the southerntier states with case reports from Kansas, Minnesota, Oregon, and Virginia; (2) reports of PAM cases following religious baptisms and nasal cleansings (ablations) worldwide; and (3) three fatal PAM cases in Louisiana over the period, 2011-2013, with deaths of two adults in 2011 whose only freshwater exposures were regular sinus irrigations with neti pots filled with tap water from municipal drinking water treatment systems and a third death in a 4-year-old child whose only freshwater exposure was playing all day on a hose-fed outdoor Slip 'n Slide. (7) Although the neti pots were negative for N. fowleri by PCR in the adult cases, tap water and swab samples taken in both households tested positive for N. fowleri. (7) In the pediatric case, the municipal water supply tested positive for N. fowleri at the household and other parish sites (Source: The New Orleans Times Picayune, September 13, 2013. Alexander-Bloch B. Deadly amoeba found in St. Bernard water).

Free-living amebae, including N. fowleri have now been isolated from several habitats worldwide including standing freshwater, thermal discharges from power plants, municipal drinking water treatment systems, potable well water, soil, sewage, and even the nasal passages and throats of patients with and without upper respiratory diseases. (8-11) Thomas and Ashbolt determined a mean free-living ameba detection rate of 45% in a 2011 meta-analysis of 26 reports of the presence of free-living amebae in drinking water treatment systems in 18 different countries. (11) N. fowleri can proliferate in domestic water supplies with low flow rates, increasing temperatures, and falling residual chlorine levels. (9, 10) Cursons and co-investigators previously determined that cysticidal residual chlorine concentrations for N. fowleri are in the range of 0.5-1.0 mg/L, but household and, especially, hot water heater, residual chlorine levels often drop well below that range as increasing temperatures lower chlorine levels. (12) In light of the latest PAM death in Louisiana, a child playing outdoors in a hose-fed Slip 'n Slide, chlorine levels in wade and swimming pools should be maintained above 1.0 mg/L. In summary, free-living amebae may be detected in nearly half of the world's drinking water treatment systems today. N. fowleri can proliferate in domestic water supplies as temperatures rise and chlorine levels fall, and residual chlorine levels must be maintained above 0.5 mg/L in drinking water supplies to remain cysticidal for N. fowleri. (8-12)

Since there are no standardized, microbiological testing methods to detect and to quantitate N. fowleri amebae in recreational freshwater, PAM is best prevented by a combination of untested and not evidence-based educational and behavioral modification strategies including the following: (1) Avoid water-related activities, such as swimming, diving, water skiing, and wakeboarding in bodies of warm freshwater, hot springs, and thermally-polluted water like that around coal-burning and nuclear electrical power plants. (2) Avoid similar water-related activities in warm freshwater during prolonged periods of high water temperatures and low water levels. (3) Hold the nose shut, use nose clips, or hold the head above water to avoid any traumatic freshwater injections with disruptions in the nasal mucosal linings during water-related activities or religious baptisms or other practices in warm freshwater, such as lakes, rivers, ponds, bayous, freshwater-filled quarries, and hot springs. (4) Avoid similar water-related activities in drainage ditches, retention or oxidation ponds, and irrigation canals. (5) Avoid digging or stirring up the bottom sediment during all water-related activities in shallow, warm freshwater bodies. (6) When irrigating the nose and nasal sinuses, always use water that has been previously boiled for one minute (boil for three minutes at elevations above 3,500 feet) and left to cool; or use chlorinated tap-water filtered with a 1-micron or smaller pore-size filter; or use bottled distilled or sterilized water. (7) (7) Always rinse neti pots or other nasal irrigating devices after each use with boiled, filtered, distilled, or sterilized water; and allow all nasal irrigation devices to air-dry completely before reuse. (7)

In 1997, Kramer and co-investigators reported successful kidney and liver transplantation from a deceased donor with laboratory-confirmed N. fowleri infection of the brain. (13) More recently, Bennett and colleagues reported procuring several organs (liver, lung, kidneys, and pancreas) from a 12-year-old male donor dying from PAM in Oregon in 2008 and transplanted the organs into several recipients without transplant-transmitted N. fowleri infections. (14) Since only the ameboid trophozoites and not the cyst or flagellated forms of N. fowleri penetrate the CNS via the nose and not by hematogenous spread from multiple granulomas of the skin and other organs in acanthamoebiasis and balamuthiasis, deceased donors with N. fowleri CNS infections may be suitable organ donors. (13,14)

Results: Granulomatous amebic encephalitis (GAE)

Granulomatous amebic encephalitis (GAE) is a chronic infection of the brain that may disseminate to other organs hematogenously and usually occurs in immunosuppressed patients with AIDS or organ transplants, or in patients receiving chemotherapy for cancer or tuberculosis. GAE may be caused by several species of Acanthamoeba or by another, phylogenetically related, free-living amebic species, Balamuthia mandrillaris. Acanthamoeba species and B. mandrillaris are distributed worldwide in freshwater and soil and can cause GAE year-round without climatic or seasonal periodicity. The portal of entry for these opportunistic pathogens is through the respiratory tract or via ulcerating skin wounds with hematogenous spread to the CNS and, less commonly, with widespread hematogenous dissemination to other organs in the severely immunocompromised.

To date, approximately 200 cases of Acanthamoeba GAE and 150 cases of Balamuthia GAE have been reported with acanthamoebiasis still confined mostly to the immunocompromised and balamuthiasis affecting both immunocompromised and immunocompetent individuals. (2) Besides immunocompromise, other potential risk factors for balamuthiasis may include contact with stagnant freshwater or with contaminated soil, often through agricultural or other occupational work, desert motorcycling, dirt-biking, or gardening. (2) There were 28 CDC-confirmed cases of B. mandrillaris GAE in the US during the reporting period, 1994-2010. (2) Of the 28 cases, most cases occurred in immunocompetent patients, with an age range of 1.5 to 89 years. The mean age of the study population was 25.04 ([+ or -] 25.85) years with males (n = 20) outnumbering females. There were no statically significant differences in the mean ages of male and female cases, and there were few survivors of B. mandrillaris GAE (n = 7, 4 males, 3 females). Primary inoculation with Balamuthia mandrillaris was typically via the skin or lungs, and the incubation periods were shorter than in Acanthamoeba GAE, with a mean of 8.5 days and a range of 1-30 days.

The microscopic diagnosis of balamuthiasis is made by the detection of cyst forms and/or trophozoites in the brain, eyes, skin, lungs, and other organs. (15) Recently, immunodiagnostic tests, such as indirect immunofluorescent ultraviolet microscopy and indirect immunofluorescent antibody ultraviolet microscopy with specific anti-pathogen antibodies, and new PCR assays for identification of pathogen DNA have been developed for diagnostic specimens. In 2006, Qvarnstrom and co-investigators at the CDC described a new multiplex real-time PCR assay for the simultaneous detection of Acanthamoeba spp., B. mandrillaris, and N. fowleri, which will permit rapid and specific detection of a single free-living ameba in clinical specimens within five hours. (16)

Neuroimaging studies by axial CT and/or MRI in GAE are nonspecific and often include single to multiple space-occupying lesions in the brain from the frontal cortex to the cerebellum with ring-enhancing and other focal effects slightly more common in balamuthiasis than acanthamoebiasis. (17)

Successful treatment strategies for GAE have included combinations of critical care management techniques to reduce increased intracranial pressure, craniotomy for biopsy or excision of mass lesions, and combination pharmacotherapy with antifungals, anti-protozoal agents, synergistic antibiotics, and several experimental therapies that have shown promise in vitro, such as the phenothiazines and miltefosine. (6) Schuster and others have now demonstrated that the phenothiazines and a variety of antibiotics, antifungals, and antiparasitics all display in vitro efficacy against B. mandrillaris in clinical specimens. (6) In 2008, Aichelburg and colleagues in Vienna reported treating a patient successfully with disseminated tuberculosis and acanthamoebiasis with topical and oral miltefosine and a combination of intravenous fluconazole, trimethoprim-sulfamethoxazole, a synergistic antibiotic (amikacin), and four tuberculostatic drugs. (18)

The incubation period for Acanthamoeba GAE could extend for weeks or months after primary inoculation in the skin, sinuses, or lungs, with subsequent draining ulcers, chronic sinusitis, or pneumonia. Acanthamoebae have been well recognized as opportunistic pathogens causing fatal GAE in patients with tuberculosis and AIDS and following organ transplants since the 1990s. In 1999, Oliva and colleagues successfully treated a patient with widely disseminated acanthamoebiasis following a single lung transplant for sarcoidosis with a combination of pentamidine, 5-fluorocytosine, itraconazole, and topical chlorhexidine gluconate-ketoconazole cream for skin lesions. (19) The patient presented with more than 20 painful nodular skin lesions scattered over the trunk and extremities, elevated liver enzymes, and respiratory failure. (19) In 2007, Barete and co-authors reported a fatal case of acanthamoebiasis - despite treatment with pentamidine, 5-fluorocytosine, and itraconazole - in a heart transplant patient who presented with carbuncles and abscesses scattered over the extremities and trunk, septic shock, and multi-organ failure. (20)

Like the Acanthamoeba species, disseminated B. mandrillaris infections also cause skin lesions and GAE and were recently associated with two case clusters of organ transplant -transmitted infections in the US, with three deaths in organ transplant recipients. (2) In late 2009, a 4-year-old boy living in Mississippi developed a transient febrile illness, was diagnosed with influenza, and treated with antivirals. (2) Within a week, he developed headache and seizures with cerebral edema and ring-enhancing intracerebral lesions on MRI, consistent with a diagnosis of post-influenza autoimmune disseminated encephalomyelitis. (2) Despite supportive treatment for seizures and cerebral edema, seizures recurred, intracranial lesions progressed, and cerebral herniation and brain death occurred approximately two weeks following the onset of CNS symptoms. (2) The child's heart, liver, and kidneys were transplanted into four different recipients at three transplant centers. (2) Later, histopathological examination of donor brain tissue at the CDC was consistent with free-living amebic encephalitis, later PCR-confirmed as B. mandrillaris GAE. (2) The donor frequently played outdoors and had confirmed soil and wading pool exposures. (2) Of the four organ transplant recipients, asymptomatic pediatric heart and liver transplant recipients were treated prophylactically for Balamuthia exposures and remained well. A 31-year-old female kidney transplant recipient died of PCR-confirmed Balamuthia GAE, despite weeks of intensive care and therapy with multiple agents known to be effective for balamuthiasis; and a 27-year-old male kidney transplant recipient recovered from PCR-confirmed Balamuthia GAE with permanent neurologic sequelae. (2)

In September 2010, the CDC reported a second PCR-confirmed cluster of organ transplant-transmitted Balamuthia GAE in Arizona. (2) The common organ donor, a 27-year-old Hispanic male landscaper with a six-month history of a non-healing skin lesion on his back attributed to an insect bite, died from a presumed stroke. (2) The donor's heart, liver, pancreas, and kidneys were transplanted into four recipients, two of whom, a kidney-pancreas recipient and a liver recipient, died of PCR-confirmed Balamuthia GAE. (2) Asymptomatic heart and kidney transplant recipients were placed on prophylactic combined antimicrobial therapy and have remained well. (2)

Untested prevention and control strategies for GAE may include (1) consideration of GAE in organ transplant and immunocompromised patients with encephalitis and skin ulcers not improving with standard therapies; (2) recognition of genetic and occupational risk factors for acanthamoebiasis and balamuthiasis in Hispanics, who may be less able to produce antibodies against causative free-living amebae; and (3) recognition of other soil or stagnant freshwater risk factors in both immunocompetent and immunosuppressed patients with granulomatous skin ulcers and unexplained meningoencephalitis.


Free-living amebae of the genera Acanthamoeba, Balamuthia, and Naegleria are typically rare causes of meningoencephalitis in humans. Amebic meningoencephalitis is often underdiagnosed and may be more common than initially reported, especially among potential organ donors with rapidly fatal meningoencephalitis of uncertain etiologies. Some forms of amebic encephalitis are capable of hematogenous dissemination from transplanted organs from infected donors to immunosuppressed organ recipients. Risk factors for PAM included male gender, freshwater exposures, summer exposures, and exposures in southern-tier US states. Risk factors for GAE included male gender, exposures in southern-tier US states, Hispanic ethnicity in California, occupational or recreational contacts with soil, and recent organ transplantation. Clinicians should suspect free-living amebic infections of the CNS in refractory cases of meningoencephalitis initially managed as aseptic or bacterial infections of the brain. In such cases, the microbial etiology of the infection should be established in brain dead donors prior to the harvesting of transplantable organs.


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(6.) Schuster FL, Guglielmo BJ, Visvesvara GS. In vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebae: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. J Eukaryot Microbiol 2006;53:121-126.

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(14.) Bennett WM, Nespral JF, Rosson MW, McEvoy KM. Use of organs for transplantation from a donor with primary meningoencephalitis due to Naegleria fowleri. Am J Transplant 2008;8:1334-1335.

(15.) Deol I, Robledo L, Maza A, Visvesvara GS, Andrews RJ. Encephalitis due to a free-living amoeba (Balamuthia mandrillaris): case report with literature review. Surg Neurol 2000;53:611-616.

(16.) Qvarnstrom Y, Visvesvara GS, Sriram R, daSilva AJ. Multiplex realtime PCR assay for simultaneous detection of Acanthamoebae spp., Balamuthia mandrillaris, and Naegleria fowleri. J Clin Microbiol 2006;44:3589-3595.

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(19.) Oliva S, Jantz M, Tiernan R, Cook DL, Judson MA. Successful treatment of widely disseminated acanthamoebiasis. So Med J 1999;92:55-57.

(20.) Barete S, Combes A, de Jonckheere JF, Datry A, Varnous S, Martinez V, et al. Fatal disseminated Acanthamoeba lenticulata infection in a heart transplant patient. Emerg Infect Dis 2007;13:736-738.

James H. Diaz, MD, DrPH, FCCM, FACPM; J. Philip Boudreaux, MD, FACS

Dr. Diaz is Professor of Public Health and Preventive Medicine and Head of Environmental and Occupational Health Sciences in the School of Public Health and Professor of Anesthesiology/Critical Care Medicine in the School of Medicine at Louisiana State University Health Sciences Center in New Orleans. Dr. Boudreaux is Professor of Surgery in the School of Medicine at LSUHSC-New Orleans.
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Author:Diaz, James H.; Boudreaux, J. Philip
Publication:The Journal of the Louisiana State Medical Society
Article Type:Report
Geographic Code:1USA
Date:Nov 1, 2013
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