Balamuthia and Acanthamoeba-binding antibodies in West African human sera.

Little is known about the prevalence of Balamuthia mandrillaris amoebae and Balamuthia amoebic encephalitis in Africa. As an approach, relative concentrations of amoebae-binding serum antibodies (Ab) were assessed by flow cytometry using formaldehyde-fixed B. mandrillaris, Acanthamoeba lenticulata 72-2 and Acanthamoeba castellanii 1BU amoebae for specific Ab capture (B.m.-Ab, A.l.-Ab, A.c.-Ab). One hundred and ninety-two sera from West African (Côte d'Ivoire) donors aged 11-95years (mean 38 a; 51% males), and living in villages surrounded by rainforest near the Liberian border, were tested and related to reference sera from Berlin. While B.m.-Ab tended to increase with donor age, A.l.-Ab and A.c.-Ab did not. Accordingly, B.m.-Ab correlated only weakly with A.l.-Ab or A.c.-Ab. Of the nine individuals with the highest B.m.-Ab concentrations, most were elderly (mean 58 a), male (78%), and professed intensive outdoor activity (hunting, farming). Only three of these sera also showed elevated A.l.-Ab, and none elevated A.c.-Ab.

Molecular confirmation of Sappinia pedata as a causative agent of amoebic encephalitis.

Pathogenic free-living amoebae, such as Acanthamoeba species, Balamuthia mandrillaris, and Naegleria fowleri, are known to cause infections of the central nervous system in human and other animals. In 2001, a case of human encephalitis was reported that was caused by another amoeba with morphological features suggestive of Sappinia. The amoeba originally identified as Sappinia diploidea was identified, most likely as S. pedata, by use of newly developed real-time polymerase chain reaction assays. This amoeba had previously been found only in environmental sources, such as soil and tree bark. The results illustrate the potential for other free-living amoebae, which are not normally associated with human disease, to cause occasional infections.

Under the radar: balamuthia amebic encephalitis.

BACKGROUND: We present data from 9 years (1999-2008) of tests for Balamuthia mandrillaris, an agent of amebic encephalitis that were conducted as part of the California Encephalitis Project. METHODS: Specimens obtained from patients with encephalitis were sent to the California Encephalitis Project for diagnostic testing; a subset of these specimens were tested for Balamuthia species. Tests included indirect immunofluorescent staining of sections for amebae, fluorescent antibody staining and enzyme-linked immunosorbent assay for serum titers, and polymerase chain reaction for Balamuthia 16S mitochondrial DNA. Cerebrospinal fluid (CSF) samples obtained from patients with diverse types of encephalitis were also tested for a broad range of cytokines. RESULTS: Of >3500 cases referred to the California Encephalitis Project, 10 were found to be amebic encephalitis on the basis of serologic and CSF tests and examination of stained tissue sections. Most of these cases would have been described as "encephalitis of unknown origin" if it were not for the California Encephalitis Project. Nine of the 10 patients were male; ages ranged from 1.5 to 72 years. All patients had abnormal neuroimaging findings and abnormal CSF composition. The more common symptoms at presentation included headache, seizures, cranial nerve palsies, and lethargy. CSF specimens from patients with Balamuthia infection had significant elevations in the levels of cytokines IL-6 and IL-8, compared with specimens obtained from persons with viral or noninfectious encephalitides. CONCLUSIONS: Balamuthiasis is difficult to diagnose, and it is likely that cases go unrecognized because clinicians and laboratorians are unfamiliar with the disease. Alerting the medical community to this disease may lead to earlier diagnosis and improve the chances of survival.

Eosinophilic meningitis due to Angiostrongylus and Gnathostoma species.

Eosinophilic meningitis can be the result of noninfectious causes and infectious agents. Among the infectious agents, Angiostrongylus cantonensis and Gnathostoma spinigerum are the most common. Although angiostrongyliasis and gnathostomiasis are not common in the United States, international travel and immigration make these diseases clinically relevant. Both A. cantonensis and G. spinigerum infection can present as severe CNS compromise. Diagnoses of both infections can be challenging and are often clinical because of a paucity of serological assays readily available in the United States. Furthermore, there are conflicting recommendations about treatment for angiostrongyliasis and gnathostomiasis. To further explore the emerging nature of these helminthic infections, a case description and review of A. cantonensis and G. spinigerum infections are presented. The clinical severity of eosinophilic meningitis and diagnosis of these infections are highlighted.

Current world status of Balantidium coli.

Balantidium coli is a cosmopolitan parasitic-opportunistic pathogen that can be found throughout the world. Pigs are its reservoir hosts, and humans become infected through direct or indirect contact with pigs. In rural areas and in some developing countries where pig and human fecal matter contaminates the water supply, there is a greater likelihood that balantidiosis may develop in humans. The infection may be subclinical in humans, as it mostly is in pigs, or may develop as a fulminant infection with bloody and mucus-containing diarrhea; this can lead to perforation of the colon. The disease responds to treatment with tetracycline or metronidazole. Balantidiosis is a disease that need never exist given access to clean water and a public health infrastructure that monitors the water supply and tracks infections. Its spread can be limited by sanitary measures and personal hygiene, but it is a disease that will be around as long as there are pigs. Immunocompromised individuals have developed balantidiosis without any direct contact with pigs, perhaps with rats or contaminated produce as a possible source of infection. For the clinician, balanatidiosis should be included in the differential diagnosis for persistent diarrhea in travelers to or from Southeast Asia, the Western Pacific islands, rural South America, or communities where close contact with domestic swine occurs. Warming of the earth's surface may provide a more favorable environment, even in the now-temperate areas of the world, for survival of trophic and cystic stages of Balantidium, and its prevalence may increase. Effective sanitation and uncontaminated water are the most useful weapons against infection. Fortunately, balantidiosis responds to antimicrobial therapy, and there have been no reports of resistance to the drugs of choice.

Balamuthia mandrillaris, agent of amebic encephalitis: detection of serum antibodies and antigenic similarity of isolates by enzyme immunoassay.

We report the development of an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Balamuthia mandrillaris, a free-living ameba that is an etiologic agent of granulomatous amebic encephalitis (GAE). As part of the California Encephalitis Project (CEP), we have tested serum and cerebrospinal fluid (CSF) samples from a subgroup of 130 hospitalized encephalitis patients (out of approximately 430 samples) over a 16-month period. Case criteria were based on clinical, laboratory, and occupational/recreational histories. All serum samples initially underwent screening by immunofluorescent antibody (IFA) staining with results ranging from no detectable ameba antibodies to titers of 1:256. In addition to the 130 samples tested prospectively, sera and/or CSF from 11 previously confirmed cases of balamuthiasis, six healthy individuals, and earlier CEP submissions with high IFA antibody titers were also tested retrospectively. Among the 130 samples, two cases of balamuthiasis were identified by ELISA and confirmed by the polymerase chain reaction (PCR). The availability of sera from human and animal cases and from varied geographic areas allowed comparisons of serologic similarities of the different Balamuthia strains and human sera. All sera, whether from human or other mammals, reacted with all strains of Balamuthia, as they did with Balamuthia amebae from different geographic areas. Enzyme-linked immunosorbent assay results were consistent with the IFA results. Differences between readings were likely due to cross-reactivity between Balamuthia antigens and unidentified antibodies in serum.

Demonstration of Balamuthia and Acanthamoeba mitochondrial DNA in sectioned archival brain and other tissues by the polymerase chain reaction.

Granulomatous amoebic encephalitis (GAE) is a usually fatal disease caused by the free-living amoebae Balamuthia mandrillaris and Acanthamoeba spp. The intent of this study was to determine if the polymerase chain reaction (PCR) could be used retrospectively to detect amoeba mitochondrial 16S rRNA gene DNA in confirmed archival tissue sections from GAE cases stored in our laboratories for 1 to 34 years. The DNA was extracted from deparaffinized sections, and appropriate primer sets for each of the two amoebae were used for amoeba DNA detection. Indirect immunofluorescent (IIF) staining of tissue sections was used as the standard for identification of amoebae against which the PCR results were compared. Sixty slides from a total of 56 cases were processed by PCR for amoeba 16S DNA. In 28 slides (47%), there was agreement between the IIF and PCR results. In 41 of the slides (52%), no amoeba DNA was detected after PCR. In one slide (1%), the PCR and IIF results did not agree. While PCR supported IIF findings in about half of the slides, there are significant limitations in amoeba DNA identifications in formalin-fixed brain tissues. Degradation of amoeba DNA caused by formalin fixation was probably a factor in limiting valid results.

Demonstration of Balamuthia and Acanthamoeba mitochondrial DNA in sectioned archival brain and other tissues by the polymerase chain reaction.

Granulomatous amoebic encephalitis (GAE) is a usually fatal disease caused by the free-living amoebae Balamuthia mandrillaris and Acanthamoeba spp. The intent of this study was to determine if the polymerase chain reaction (PCR) could be used retrospectively to detect amoeba mitochondrial 16S ribosomal ribonucleic acid gene deoxyribonucleic acid (DNA) in confirmed archival tissue sections from GAE cases stored in our laboratories for 1 to 34 years. The DNA was extracted from deparaffinized sections, and appropriate primer sets for each of the two amoebae were used for DNA detection. Indirect immunofluorescent staining (IIF) of tissue sections was used as the standard for identification of amoebae against which the PCR results were compared. Sixty slides from a total of 56 cases were processed by PCR for amoeba 16S DNA. In 28 (47%) slides, there was agreement between the IIF and PCR results. In 41 of the slides (52%), no DNA was detected after PCR. In one slide (1%), the PCR and IIF results did not agree. While PCR supported IIF findings in about half of the slides, there are significant limitations in amoeba DNA identifications in formalin-fixed brain tissues. Degradation of amoeba DNA because of formalin fixation was probably a factor in limiting valid results.

Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea.

Among the many genera of free-living amoebae that exist in nature, members of only four genera have an association with human disease: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri and Sappinia diploidea. Acanthamoeba spp. and B. mandrillaris are opportunistic pathogens causing infections of the central nervous system, lungs, sinuses and skin, mostly in immunocompromised humans. Balamuthia is also associated with disease in immunocompetent children, and Acanthamoeba spp. cause a sight-threatening infection, Acanthamoeba keratitis, mostly in contact-lens wearers. Of more than 30 species of Naegleria, only one species, N. fowleri, causes an acute and fulminating meningoencephalitis in immunocompetent children and young adults. In addition to human infections, Acanthamoeba, Balamuthia and Naegleria can cause central nervous system infections in animals. Because only one human case of encephalitis caused by Sappinia diploidea is known, generalizations about the organism as an agent of disease are premature. In this review we summarize what is known of these free-living amoebae, focusing on their biology, ecology, types of disease and diagnostic methods. We also discuss the clinical profiles, mechanisms of pathogenesis, pathophysiology, immunology, antimicrobial sensitivity and molecular characteristics of these amoebae.

Diagnosis of first case of Balamuthia amoebic encephalitis in Portugal by immunofluorescence and PCR.

We report here the first Portuguese case of acute fatal granulomatous encephalitis attributed to Balamuthia mandrillaris, initially thought to be a brain tumor, which had a progressive and fatal outcome. Balamuthia mandrillaris is a free-living amoeba recognized as an uncommon agent of granulomatous encephalitis. Infections have been identified in immunocompromised hosts and in immunocompetent pediatric patients. Balamuthia infections are very rare, with only two reported cases in Europe. The case presented here occurred in a previously healthy boy who died 5 weeks after the onset of the symptoms. No evidence of immunological deficiency was noted, and testing for human immunodeficiency virus antibodies was negative. The symptoms were initially thought to be the result of a tumor, but histopathologic examination showed evidence of amoebic infection. Immunofluorescence staining of brain tissue identified B. mandrillaris as the infectious agent. The diagnosis was confirmed with PCR by detecting Balamuthia DNA in formalin-fixed brain tissue sections. Despite initiation of empirical antimicrobial therapy for balamuthiasis, the patient died 3 weeks after being admitted to the hospital. No source of infection was readily apparent.

Acanthamoeba encephalitis in patient with systemic lupus, India.

We report a fatal case of encephalitis caused by Acanthamoeba in a 24-year-old woman from India with systemic lupus erythematosus. Diagnosis was made by identification of amebas in brain sections by immunofluorescence analysis and confirmed by demonstrating Acanthamoeba mitochondrial 16S rRNA gene DNA in brain tissue sections.

Detection of antibodies against free-living amoebae Balamuthia mandrillaris and Acanthamoeba species in a population of patients with encephalitis.

BACKGROUND: Balamuthia mandrillaris and Acanthamoeba species are 2 free-living amoebae responsible for granulomatous amoebic encephalitis in humans and animals. We have screened serum samples from hospitalized patients with encephalitis for antibodies against these 2 amoebae as a means of detecting a disease with few defining symptoms and a poor prognosis. METHODS: Indirect immunofluorescence antibody (IFA) staining of serum samples from patients with encephalitis was conducted over a period of 6 years to detect amoeba antibodies. More than 250 serum samples from patients hospitalized with encephalitis were screened. Most of the samples were from patients in California and were screened as part of the California Encephalitis Project, with a small number of specimens from other states. RESULTS: During the course of the study, 7 cases of Balamuthia encephalitis were detected; all cases were detected in Hispanic individuals, and all cases were fatal. Examination of hematoxylin-eosin-stained and immunostained sections of brain tissue obtained at biopsy or autopsy for amoebae confirmed balamuthiasis in all serum samples with positive IFA results. One case of Acanthamoeba encephalitis was detected in an immunocompromised individual with a normal antibody titer by identification of amoebae in immunostained brain tissue obtained at autopsy. CONCLUSIONS: IFA can be successfully used in screening for balamuthiasis and acanthamoebiasis in patients whose clinical presentation, laboratory results, and neuroimaging findings are suggestive of amoebic encephalitis. Ideally, this can lead to an earlier definitive diagnosis and earlier start of antimicrobial therapy. Without IFA staining, the balamuthiasis cases in our study would have been diagnosed as neurocysticercosis, tumor, tuberculosis, or viral encephalitis or would have been undiagnosed.

In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri.

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.

Morphologic and molecular identification of Naegleria dunnebackei n. sp. isolated from a water sample.

Naegleria dunnebackei n. sp., a new species of the free-living amoeboflagellate Naegleria, is described in this report. The organism was isolated from a water sample taken from drinking troughs associated with cases of primary amoebic meningoencephalitis in cattle at a ranch in southern California. The isolate grew at, but not above 37 degrees C, and did not kill young mice upon intranasal inoculation suggesting that it was not pathogenic. The new species combines morphological features of non-pathogenic Naegleria gruberi and pathogenic Naegleria fowleri. The trophic amoeba resembled other members of the genus, with a prominent vesicular nucleus and mitochondria with discoidal cristae; a Golgi apparatus was not observed by electron microscopy. The cyst stage had pores in the wall typical of those seen in pathogenic N. fowleri. Upon suspension in distilled water, amoebae transformed into temporary, non-feeding flagellates, mostly with two anterior flagella but occasionally with four. The rationale for its description as a new species was based upon sequencing of the 5.8S rDNA and internal transcribed spacers of the amoeba, which is similar to but not identical to that of Naegleria gallica, differing from that organism's DNA by six base pairs. Virus-like elements were found in the cytoplasm of trophic amoebae, often in association with crystalloids, and may be the cause of lysis of amoebae in culture.

Detection of Balamuthia mitochondrial 16S rRNA gene DNA in clinical specimens by PCR.

Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in both immunocompromised and immunocompetent individuals. Because of a lack of pathognomonic symptoms and the difficulty in recognizing amebas in biopsied tissues, most cases are not diagnosed or effectively treated, leading to a >95% mortality. We report here on five cases of balamuthiasis that were diagnosed by indirect immunofluorescence (IIF) staining of serum for anti-Balamuthia antibodies (titer > or = 1:128) and confirmed by IIF of unstained brain tissue sections and/or detection of amebas in hematoxylin-eosin-stained slides. Additionally, we have used the PCR for the detection of mitochondrial 16S rRNA gene DNA from the ameba in clinical specimens such as brain tissue and cerebrospinal fluid (CSF) from individuals with Balamuthia encephalitis. Balamuthia DNA was successfully detected by the PCR in clinical samples from all five individuals. It was detected in brain tissue from three cases, in CSF from three cases, and in one of two samples of lung tissue from two individuals, but not in two samples of kidney tissue tested. One sample of unfixed brain tissue was culture positive for Balamuthia. In order to test the sensitivity of the PCR for detection of Balamuthia DNA, CSF specimens from two individuals negative for amebic infection were spiked with Balamuthia amebas. We found that it was possible to detect Balamuthia DNA in the PCR mixtures containing mitochondrial DNA from 1 to as little as 0.2 ameba per reaction mixture. A single Balamuthia ameba contains multiple mitochondrial targets; thus, 0.2 ameba represents multiple targets for amplification and is not equivalent to 0.2 of an ameba as a target.

Inability to make a premortem diagnosis of Acanthamoeba species infection in a patient with fatal granulomatous amebic encephalitis.

Granulomatous amebic encephalitis (GAE), an infection of immunocompromised hosts, is almost uniformly fatal. A case of GAE in a patient who failed to mount a serologic response to Acanthamoeba polyphaga is presented. Although Acanthamoeba polyphaga that is sensitive to multiple antimicrobials grew from brain tissue, an inability to make a premortem diagnosis precluded therapy.

Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease.

The roles free-living amebae and the parasitic protozoa Entamoeba histolytica and Balantidium coli play as agents of waterborne zoonotic diseases are examined. The free-living soil and water amebae Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are recognized etiologic agents of mostly fatal amebic encephalitides in humans and other animals, with immunocompromised and immunocompetent hosts among the victims. Acanthamoeba spp. are also agents of amebic keratitis. Infection is through the respiratory tract, breaks in the skin, or by uptake of water into the nostrils, with spread to the central nervous system. E. histolytica and B. coli are parasitic protozoa that cause amebic dysentery and balantidiasis, respectively. Both intestinal infections are spread via a fecal-oral route, with cysts as the infective stage. Although the amebic encephalitides can be acquired by contact with water, they are not, strictly speaking, waterborne diseases and are not transmitted to humans from animals. Non-human primates and swine are reservoirs for E. histolytica and B. coli, and the diseases they cause are acquired from cysts, usually in sewage-contaminated water. Amebic dysentery and balantidiasis are examples of zoonotic waterborne infections, though human-to-human transmission can occur. The epidemiology of the diseases is examined, as are diagnostic procedures, anti-microbial interventions, and the influence of globalization, climate change, and technological advances on their spread.

Balamuthia mandrillaris from soil samples.

Balamuthia mandrillaris amoebas are recognized as a causative agent of granulomatous amoebic encephalitis, a disease that is usually fatal. They were first recognized when isolated from the brain of a mandrill baboon that died in the San Diego Zoo Wild Life Animal Park. Subsequently, the amoebas have been found in a variety of animals, including humans (young and old, immunocompromised and immunocompetent persons), in countries around the world. Until recently, the amoebas had not been recovered from the environment and their free-living status was in question. The recovery of a Balamuthia amoeba from a soil sample taken from a plant at the home of a child from California, USA, who died of Balamuthia amoebic encephalitis, was reported previously. In a continued investigation, a second amoeba was isolated from soil that was obtained from an outdoor potted plant in a spatially unrelated location. A comparison of these two environmental amoebas that were isolated from different soils with the amoeba that was obtained from the child's clinical specimen is reported here. Included are the isolation procedure for the amoebas, their growth requirements, their immunological response to anti-Balamuthia serum, their sensitivity to a selection of antimicrobials and sequence analysis of their 16S rRNA gene. The evidence is consistent that the amoebas isolated from both soil samples and the clinical isolate obtained from the Californian child are B. mandrillaris.