Transmission of Balamuthia mandrillaris through solid organ transplantation: utility of organ recipient serology to guide clinical management.

A liver, heart, iliac vessel and two kidneys were recovered from a 39-year-old man who died of traumatic head injury and were transplanted into five recipients. The liver recipient 18 days posttransplantation presented with headache, ataxia and fever, followed by rapid neurologic decline and death. Diagnosis of granulomatous amebic encephalitis was made on autopsy. Balamuthia mandrillaris infection was confirmed with immunohistochemical and polymerase chain reaction (PCR) assays. Donor and recipients' sera were tested for B. mandrillaris antibodies. Donor brain was negative for Balamuthia by immunohistochemistry and PCR; donor serum Balamuthia antibody titer was positive (1:64). Antibody titers in all recipients were positive (range, 1:64-1:512). Recipients received a four- to five-drug combination of miltefosine or pentamidine, azithromycin, albendazole, sulfadiazine and fluconazole. Nausea, vomiting, elevated liver transaminases and renal insufficiency were common. All other recipients survived and have remained asymptomatic 24 months posttransplant. This is the third donor-derived Balamuthia infection cluster described in solid organ transplant recipients in the United States. As Balamuthia serologic testing is only available through a national reference laboratory, it is not feasible for donor screening, but may be useful to determine exposure status in recipients and to help guide chemotherapy.

Risk for transmission of Naegleria fowleri from solid organ transplantation.

Primary amebic meningoencephalitis (PAM) caused by the free-living ameba (FLA) Naegleria fowleri is a rare but rapidly fatal disease of the central nervous system (CNS) affecting predominantly young, previously healthy persons. No effective chemotherapeutic prophylaxis or treatment has been identified. Recently, three transplant-associated clusters of encephalitis caused by another FLA, Balamuthia mandrillaris, have occurred, prompting questions regarding the suitability of extra-CNS solid organ transplantation from donors with PAM. During 1995-2012, 21 transplant recipients of solid organs donated by five patients with fatal cases of PAM were reported in the United States. None of the recipients developed PAM, and several recipients tested negative for N. fowleri by serology. However, historical PAM case reports and animal experiments with N. fowleri, combined with new postmortem findings from four patients with PAM, suggest that extra-CNS dissemination of N. fowleri can occur and might pose a risk for disease transmission via transplantation. The risks of transplantation with an organ possibly harboring N. fowleri should be carefully weighed for each individual recipient against the potentially greater risk of delaying transplantation while waiting for another suitable organ. In this article, we present a case series and review existing data to inform such risk assessments.

Fulminant and fatal encephalitis caused by Acanthamoeba in a kidney transplant recipient: case report and literature review.

Acanthamoeba is the most common cause of granulomatous amebic encephalitis, a typically fatal condition that is classically described as indolent and slowly progressive. We report a case of Acanthamoeba encephalitis in a kidney transplant recipient that progressed to death within 3 days of symptom onset and was diagnosed at autopsy. We also review clinical characteristics, treatments, and outcomes of all published cases of Acanthamoeba encephalitis in solid organ transplant (SOT) recipients. Ten cases were identified, and the infection was fatal in 9 of these cases. In 6 patients, Acanthamoeba presented in a fulminant manner and death occurred within 2 weeks after the onset of neurologic symptoms. These acute presentations are likely related to immunodeficiencies associated with solid organ transplantation that result in an inability to control Acanthamoeba proliferation. Skin lesions may predate neurologic involvement and provide an opportunity for early diagnosis and treatment. Acanthamoeba is an under-recognized cause of encephalitis in SOT recipients and often presents in a fulminant manner in this population. Increased awareness of this disease and its clinical manifestations is essential to attain an early diagnosis and provide the best chance of cure.

Molecular characterization of Acanthamoeba isolated in water treatment plants and comparison with clinical isolates.

A total of 116 samples (44 clinical specimens and 72 environmental samples) have been analyzed for the presence of Acanthamoeba. The environmental samples (ESs) were collected from four drinking water treatment plants (DWTP, n=32), seven wastewater treatment plants (n=28), and six locations of influence (n=12) on four river basins from the central area of Spain (winter-spring 2008). Water samples were concentrated by using the IDEXX Filta-Max(®) system. Acanthamoeba was identified in 65 of the 72 ESs by culture isolation (90.3%) and 63 by real-time PCR (87.5%), resulting in all sampling points (100%) positive for Acanthamoeba when considering both techniques and all the time period analyzed. Nine of the 44 clinical specimens were positive for Acanthamoeba. Seventeen Acanthamoeba strains (eight from four DWTP and nine from clinical samples) were also established in axenic-PYG medium. Twenty-four of the ESs and the 17 Acanthamoeba sp. strains were genotyped as T4/1, T4/8, and T4/9. The eight strains isolated from the DWTP samples were inoculated in nude mouse to ascertain their potential pathogenicity in this model. Animals that were inoculated died or showed central nervous system symptoms 9 days post-inoculation. Examination of immunofluorescence-stained brain and lung tissue sections showed multiple organisms invading both tissues, and re-isolation of throphozoites was successful in these tissues of all infected animals. For the first time, potentially pathogenic Acanthamoeba T4 has been detected in 100% of different types of water samples including tap water and sewage effluents in the central area of Spain suggesting a potential health threat for humans especially for the contact lens wearers.

The epidemiology of primary amoebic meningoencephalitis in the USA, 1962-2008.

Naegleria fowleri, a free-living, thermophilic amoeba ubiquitous in the environment, causes primary amoebic meningoencephalitis (PAM), a rare but nearly always fatal disease of the central nervous system. While case reports of PAM have been documented worldwide, very few individuals have been diagnosed with PAM despite the vast number of people who have contact with fresh water where N. fowleri may be present. In the USA, 111 PAM case-patients have been prospectively diagnosed, reported, and verified by state health officials since 1962. Consistent with the literature, case reports reveal that N. fowleri infections occur primarily in previously healthy young males exposed to warm recreational waters, especially lakes and ponds, in warm-weather locations during summer months. The annual number of PAM case reports varied, but does not appear to be increasing over time. Because PAM is a rare disease, it is challenging to understand the environmental and host-specific factors associated with infection in order to develop science-based, risk reduction messages for swimmers.

Diagnostic exercise. Cerebral mass in a puppy with respiratory distress and progressive neurologic signs.

A 5-month-old mongrel puppy with a history of respiratory disease presented with progressive neurologic dysfunction. Hematologic results included leukocytosis (neutrophilia with a left shift) and lymphopenia. A mass in the right forebrain, identified by magnetic resonance imaging, was biopsied during decompressive craniectomy. The histologic diagnosis was granulomatous meningoencephalitis with intralesional amoebae. The dog died within 24 hours of surgery. At necropsy, a well-demarcated granuloma was confined to the cerebrum, but granulomatous pneumonia was disseminated through all lobes of the lung. Concurrent infections included canine distemper, canine adenoviral bronchiolitis, and oral candidiasis. Canine distemper virus probably caused immunosuppression and increased susceptibility to secondary infections.

Pathogenic amoebas from brackish and ocean sediments, with a description of Acanthamoeba hatchetti, n. sp.

Acanthamoeba culbertsoni was isolated from a sewage-spoil dump site near Ambrose Light, New York Bight. A second species, Acanthamoeba hatchetti, n. sp., was isolated from Brewerton Channel, Baltimore Harbor, Maryland. Both species killed laboratory mice after infection by the intranasal route.

Acanthamoeba infection in a patient with chronic graft-versus-host disease occurring during treatment with voriconazole.

We report a case of disseminated infection with Acanthamoeba in a patient with graft-versus-host disease after hematopoietic stem cell transplant (HSCT) for acute lymphocytic leukemia. The infection involved the brain, skin, and lungs and occurred despite treatment with voriconazole for mold prophylaxis, and did not respond to treatment with multiple other agents reported to have activity against Acanthamoeba. To our knowledge, infection with Acanthamoeba has been reported in 4 other patients after HSCT or bone marrow transplant, and our case is the first to be diagnosed ante-mortem.

Naegleria fowleri-associated encephalitis in a cow from Costa Rica.

Species of Naegleria, Acanthamoeba, and Balamuthia are soil amoebae that can cause encephalitis in animals and humans. Of these, Naegleria fowleri is the cause of often fatal primary meningoencephalitis in humans. N. fowleri-associated encephalitis was diagnosed in a cow that was suspected to have rabies. Only formalin-fixed brain was available for diagnosis. There was severe meningoencephalitis involving all parts of the brain and numerous amoebic trophozoites were present in lesions. The amoebae reacted with N. fowleri-specific polyclonal antibodies in an indirect immunofluorescent antibody test. This is the first report of amoebic encephalitis in any host from Costa Rica.

Disseminated Acanthamoeba sp. infection in a dog.

Several species of free-living amoebae can cause encephalomyelitis in animals and humans. Disseminated acanthamoebiasis was diagnosed in pyogranulomatous lesions in brain, thyroid, pancreas, heart, lymph nodes, and kidney of a one-year-old dog. Acanthamoeba sp. was identified in canine tissues by conventional histology, by immunofluorescence, by cultivation of the parasite from the brain of the dog that had been stored at -70 degrees C for two months, and by PCR. The sequence obtained from the PCR product from the amoeba from the dog was compared to other sequences in the Acanthamoeba sp. ribosomal DNA database and was determined to be genotype T1, associated with other isolates of Acanthamoeba obtained from granulomatous amebic encephalitis infections in humans.

Animal model Balamuthia mandrillaris CNS infection: contrast and comparison in immunodeficient and immunocompetent mice: a murine model of "granulomatous" amebic encephalitis.

Balamuthia mandrillaris and several species of Acanthamoeba are pathogenic "opportunistic" free-living amebas which cause granulomatous encephalitis (GAE) in humans and animals. The granulomatous component is negligible or absent, particularly in immunocompromised individuals. GAE is an "opportunistic" infection, usually seen in debilitated, malnourished individuals, in patients undergoing immunosuppressive therapy for organ transplants, and in Acquired Immunodeficiency Syndrome (AIDS). From around the world 156 cases of GAE have been reported from 1956 through October 1, 1995, 59 (26 in the USA) of them caused by B. mandrillaris, at least seven of them in AIDS patients. The present study was designed to compare and contrast the susceptibility of infection, the rate of infectivity and the histopathological changes within the CNS between the mutant, severe combined immunodeficient mice (SCID) infected with B. mandrillaris and the normal immunocompetent BALB-C mice. The SCID mouse is severely deficient in B and T lymphocytes, therefore lacking immunoglobulin and cell-mediated immunity. This mouse is also prone to develop early T cell lymphomas. One thousand amebic trophozoites and cysts of B. mandrillaris were intranasally and intraperitoneally inoculated in both strains in mice. Seventy percent of the intranasally inoculated SCID mice died due to CNS infection. Amebic trophozoites and cysts were found within CNS parenchyma without inflammatory response. Death occurred from 2 to 4 weeks after inoculation. By contrast only 10 percent of the intranasally inoculated BALB-C mice died with CNS infection showing the characteristic features of GAE. None of the intraperitoneally inoculated mice developed amebic infection. The SCID and BALB-C mice were logical models to study the structural alterations within the CNS of B. mandrillaris infection. This animal model recapitulates with excellent degree of fidelity several aspects of the pathogenesis and histopathological features of free-living amebic infection in human beings.

Free-living, amphizoic and opportunistic amebas.

Amebas belonging to the genera Naegleria, Acanthamoeba and Balamuthia are free-living, amphizoic and opportunistic protozoa that are ubiquitous in nature. These amebas are found in soil, water and air samples from all over the world. Human infection due to these amebas involving brain, skin, lung and eyes has increased significantly during the last 10 years. The epidemiology, immunology, protozoology, pathology, and clinical features of the infections produced by these protozoa differ strikingly. Infection by the pathogenic Naegleria fowleri is acquired by exposure to polluted water in ponds, swimming pools and man-made lakes. Raised temperatures during the hot summer months or warm water from power plants facilitate the growth of N. fowleri. N. fowleri is a thermophilic ameba that grows well in tropical and subtropical climates. The CNS infection, called Primary Amebic Meningoencephalitis (PAM), produced by N. fowleri is characterized by an acute fulminant meningoencephalitis leading to death 3-7 days after exposure. Victims are healthy, young individuals with a history of recent water-related sport activities. The portal of entry is the olfactory neuroepithelium. The pathologic changes are an acute hemorrhagic necrotizing meningoencephalitis with modest purulent exudate, mainly at the base of the brain, brain-stem and cerebellum. Trophozoites can be seen within the CNS lesions located mainly around blood vessels. Thus far 179 cases have been reported; 81 in the USA alone. Balamuthia mandrillaris and several species of Acanthamoeba are pathogenic "opportunistic" free-living amebas which cause Granulomatous Amebic Encephalitis (GAE) in humans and animals. GAE is an infection, usually seen in debilitated, malnourished individuals, in patients undergoing immunosuppressive therapy for organ transplants and in Acquired Immunodeficiency Syndrome (AIDS). The granulomatous component is negligible, particularly in immunocompromised individuals. Pathologically these amebas produce a patchy, chronic or subacute granulomatous encephalitis with the presence of trophozoites and cysts. The portal of entry is probably through the respiratory tract or an ulceration of the skin reaching the CNS by hematogenous spread. As of October 1, 1996, 166 cases (103 due to Acanthamoeba and 63 due to Balamuthia) of GAE have been reported from around the world. Of these 103 cases due to Acanthamoeba (72 have been reported in the USA alone, > 50 in AIDS). It is well known that several species of Acanthamoeba can also produce, chronic sight threatening ulceration of the cornea called Acanthamoeba keratitis (AK), mostly in contact lens wearers or in individuals with minor corneal abrasions. Hundreds of cases of AK have been documented world wide.

Comparisons of ribosomal RNA sequences from amitochondrial protozoa: implications for processing, mRNA binding and paromomycin susceptibility.

The amitochondrial (a-mt) protozoa include four groups of organisms that are of interest as important human parasites and as probable descendents of the earliest branches of eukaryotic evolution. These organisms have not been directly compared in terms of structure and function of a specific molecule. We sequenced portions of their rRNA-encoding genes coding for the internal transcribed spacers (ITS1 and 2) and adjoining small subunit (SS), 5.8S and large subunit (LS) rRNAs. Included are sites for RNA processing, mRNA interaction and aminoglycoside binding, as well as potential protein-encoding genes. The ITS of all a-mt protozoa examined are relatively short, but otherwise diverse. They include one or two predominant nucleotides (A in Entamoeba and Trichomonas, T in Encephalitozoon and C in Giardia) and have minimal potential secondary structure, which may form the basis for the preferential processing of ITS sequences. The mechanism employed by a-mt protozoa to bind mRNA may be unique, since Giardia, Trichomonas and Entamoeba mRNAs have usually short 5' non-coding regions. In bacteria, the 3' terminus of the SS rRNA is involved in mRNA binding; analysis of Entamoeba and Trichomonas mRNA 5' non-coding sequences suggests an analogous mechanism involving potential base pairing to the loop of the terminal SS rRNA hairpin. Giardia sensitivity to paromomycin was previously correlated with the presence of a C:G bp near the decoding region of SS rRNA. This bp is also present in Entamoeba and Trichomonas, consistent with their susceptibility. Its absence in Encephalitozoon and other microsporidia predicts paromomycin resistance, and suggests a distinct evolutionary origin for this group.

Granulomatous amebic encephalitis in an AIDS patient.

A 39-year-old man with AIDS died after developing a variety of neurologic symptoms and signs. CT showed multiple enhancing lesions in the cerebral hemispheres and cerebellum. Postmortem examination revealed parenchymal hemorrhagic and necrotizing lesions with a thrombo-occlusive vasculitis due to Acanthamoeba, which was typed as Acanthamoeba group 2, probably A rhysodes, by immunofluorescence.

Respiratory epithelial cells masquerading as agents of primary amebic meningoencephalitis: distinguishing features.

CSF does not normally contain motile cells within flagella or cilia. When motile cells are seen in the CSF, they may be incorrectly assumed to be amebae. We studied a patient with a traumatic mucocele, in whom motile ciliated respiratory epithelial cells were seen in the CSF and within a frontal lobe brain abscess.

Susceptibility of Acanthamoeba to soft contact lens disinfection systems.

Members of the genus Acanthamoeba are increasingly recognized as agents of indolent, chronic, infectious keratitis. Recently, Acanthamoeba corneal infection has been reported in some persons who wear soft contact lenses. In this study, three "heat" and three "cold" soft contact lens disinfection systems were tested according to the manufacturers' instructions against Acanthamoeba castellanii and Acanthamoeba polyphaga in separate trials, and with appropriate controls. Suspensions of Acanthamoeba cysts or trophozoites of each species were tested individually. Each of the three heat disinfection units killed all acanthamoebae in one cycle in all trials. A chlorhexidine 0.005%/thimerosal 0.001% solution killed A. castellanii trophozoites and cysts, but those of A. polyphaga survived. Trophozoites and cysts of both species survived an alkyl triethanol ammonium chloride 0.013%/thimerosal 0.002% solution and a hydrogen peroxide 3% preparation. Heat disinfection overall appears to be more effective in killing Acanthamoeba trophozoites and cysts as compared to cold disinfection methods.

Pathology of symptomatic microsporidial (Encephalitozoon hellem) bronchiolitis in the acquired immunodeficiency syndrome: a new respiratory pathogen diagnosed from lung biopsy, bronchoalveolar lavage, sputum, and tissue culture.

Encephalitozoon hellem is a recently described microsporidian associated with an expanding spectrum of clinical presentations in patients with the acquired immunodeficiency syndrome (AIDS). It is morphologically similar to Encephalitozoon cuniculi, a microsporidian infection of mammals and some avians, and their differentiation rests on biochemical and antigenic analyses. This report describes a patient previously diagnosed with keratoconjunctivitis due to E hellem who subsequently was found to have respiratory tract microsporidiosis by sputum cytology. He subsequently developed pulmonary symptoms and a left lower lobe interstitial infiltrate. A bronchoalveolar lavage and transbronchial biopsy revealed microsporidial bronchiolitis, and the etiologic agent was identified as E hellem using an immunofluorescent antibody technique. Lavage fluid was successfully cultured in monkey kidney cells, and cultivated E hellem organisms were studied using immunohistochemistry as well as scanning and transmission electron microscopy. The pathologic features of this newly described cause of protozoal bronchiolitis, the role of immunofluorescent antibody examination and in vitro tissue culture for species-specific diagnosis, and the significance of microsporidial pulmonary infections in AIDS patients are discussed.

Rapid diagnosis of Acanthamoeba keratitis from corneal scrapings using indirect fluorescent antibody staining.

Two soft contact lens wearers using a homemade saline solution developed corneal stromal inflammation and epithelial ulceration and were both treated for months with a presumptive diagnosis of herpes simplex keratitis. Subsequently, corneal scrapings revealed refractile, cystic structures consistent with the appearance of Acanthamoeba. This was rapidly confirmed by indirect fluorescent antibody studies, and Acanthamoeba castellani was later identified by growth in culture in both cases. Acanthamoeba is being reported with increasing frequency as a pathogen responsible for chronic stromal keratitis and ulceration in contact lens wearers. Since specific therapy is required to control this organism, rapid diagnosis is essential. Indirect fluorescent antibody staining of corneal scrapings provides a simple means of accomplishing this goal with a high degree of accuracy.

Acanthamoeba keratitis. Potential role for topical clotrimazole in combination chemotherapy.

Clotrimazole is an antifungal agent that has been shown to have excellent in vitro activity against most strains of Acanthamoeba. We encountered four patients who developed Acanthamoeba keratitis while wearing contact lenses that had been stored in homemade saline. Their medical treatment regimens included the use of topical 1% clotrimazole. In two patients in whom conventional therapy failed, clotrimazole was successful in controlling recurrent infection following penetrating keratoplasty. Two other patients were treated with clotrimazole as well as propamidine isethionate and neomycin sulfate-polymyxin B sulfate-gramicidin from the outset, and had an excellent response to medical therapy. In those patients who found the commercially available cream uncomfortable, a 1% clotrimazole suspension formulated in artificial tears was used and found to be well tolerated.

Susceptibility of Acanthamoeba to cryotherapeutic method.

Acanthamoeba keratitis is becoming an increasingly well-known clinical entity. The Acanthamoeba species in their encysted state are resistant to antimicrobial agents, resulting in corneal infections that are refractory to medical therapy. Corneal cryotherapy has been used to treat Acanthamoeba keratitis with varying and equivocal success. We subjected trophozoites and cysts of two species of Acanthamoeba recovered from corneal infections to trials of in vitro freeze-thaw-refreeze cryotherapeutic methods. The trophozoites of both species were killed in all trials. However, the cysts of both species survived all trials. Cryotherapy may not be an effective means to eliminate Acanthamoeba cysts from the cornea.