Minimal Cerebrospinal Fluid Concentration of Miltefosine despite Therapeutic Plasma Levels during the Treatment of Amebic Encephalitis.

Miltefosine is an alkylphosphocholine compound that is used primarily for treatment of leishmaniasis and demonstrates in vitro and in vivo antiamebic activity against Acanthamoeba species. Recommendations for treatment of amebic encephalitis generally include miltefosine therapy. Data indicate that treatment with an amebicidal concentration of at least 16 µg/ml of miltefosine is required for most Acanthamoeba species. Although there is a high level of mortality associated with amebic encephalitis, a paucity of data regarding miltefosine levels in plasma and cerebrospinal fluid in vivo exists in the literature. We found that despite aggressive dosing (oral miltefosine 50 mg every 6 h) and therapeutic plasma levels, the miltefosine concentration in cerebrospinal fluid was negligible in a patient with AIDS and Acanthamoeba encephalitis.

Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing.

BACKGROUND: Primary amoebic meningoencephalitis (PAM) is a rare, often lethal, cause of encephalitis, for which early diagnosis and prompt initiation of combination antimicrobials may improve clinical outcomes. METHODS: In this study, we sequenced a full draft assembly of the Balamuthia mandrillaris genome (44.2 Mb in size) from a rare survivor of PAM, and recovered the mitochondrial genome from six additional Balamuthia strains. We also used unbiased metagenomic next-generation sequencing (NGS) and SURPI bioinformatics analysis to diagnose an ultimately fatal case of Balamuthia mandrillaris encephalitis in a 15-year-old girl. RESULTS AND DISCUSSION: Comparative analysis of the mitochondrial genome and high-copy number genes from six additional Balamuthia mandrillaris strains demonstrated remarkable sequence variation, and the closest Balamuthia homologs corresponded to other amoebae, hydroids, algae, slime molds, and peat moss. Real-time NGS testing of hospital day 6 CSF and brain biopsy samples detected Balamuthia on the basis of high-quality hits to 16S and 18S ribosomal RNA sequences present in the National Center for Biotechnology Information (NCBI) nt reference database. The presumptive diagnosis of PAM by visualization of amoebae on brain biopsy histopathology and NGS analysis was subsequently confirmed at the US Centers for Disease Control and Prevention (CDC) using a Balamuthia-specific PCR assay. Retrospective analysis of a day 1 CSF sample revealed that more timely identification of Balamuthia by metagenomic NGS, potentially resulting in a better clinical outcome, would have required availability of the complete genome sequence. CONCLUSIONS: These results underscore the diverse evolutionary origins of Balamuthia mandrillaris, provide new targets for diagnostic assay development, and will facilitate further investigations of the biology and pathogenesis of this eukaryotic pathogen. The failure to identify PAM from a day 1 sample without a fully sequenced Balamuthia genome in the database highlights the critical importance of whole-genome reference sequences for microbial detection by metagenomic NGS.

Diagnosing Balamuthia mandrillaris Encephalitis With Metagenomic Deep Sequencing.

OBJECTIVE: Identification of a particular cause of meningoencephalitis can be challenging owing to the myriad bacteria, viruses, fungi, and parasites that can produce overlapping clinical phenotypes, frequently delaying diagnosis and therapy. Metagenomic deep sequencing (MDS) approaches to infectious disease diagnostics are known for their ability to identify unusual or novel viruses and thus are well suited for investigating possible etiologies of meningoencephalitis. METHODS: We present the case of a 74-year-old woman with endophthalmitis followed by meningoencephalitis. MDS of her cerebrospinal fluid (CSF) was performed to identify an infectious agent. RESULTS: Sequences aligning to Balamuthia mandrillaris ribosomal RNA genes were identified in the CSF by MDS. Polymerase chain reaction subsequently confirmed the presence of B. mandrillaris in CSF, brain tissue, and vitreous fluid from the patient's infected eye. B. mandrillaris serology and immunohistochemistry for free-living amoebas on the brain biopsy tissue were positive. INTERPRETATION: The diagnosis was made using MDS after the patient had been hospitalized for several weeks and subjected to costly and invasive testing. MDS is a powerful diagnostic tool with the potential for rapid and unbiased pathogen identification leading to early therapeutic targeting.

Detection of biomarkers of pathogenic Naegleria fowleri through mass spectrometry and proteomics.

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry MALDI-TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF-TOF instrument. MALDI-TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI-TOF MS fingerprinting is a rapid, reproducible, high-throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.

Identification of peptidases in highly pathogenic vs. weakly pathogenic Naegleria fowleri amebae.

Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse-passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba-CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.

Successful treatment of granulomatous amoebic encephalitis with combination antimicrobial therapy.

Granulomatous amoebic encephalitis (GAE) is a rare but fatal infection. Due to its nonspecific symptoms and laboratory and neuroradiological findings, it is rarely diagnosed antemortem. We herein present the case of a 72-year-old Japanese woman who was diagnosed with GAE following the detection of a pathogen similar to Balamuthia mandrillaris under a microscopic examination of cerebrospinal fluid sediment and who achieved remission with combination antimicrobial therapy. There are no previous reports of pathogens similar to B. mandrillaris being detected in cerebrospinal fluid antemortem; therefore, this case may be used as a benchmark for further studies.

Fatal Naegleria fowleri infection acquired in Minnesota: possible expanded range of a deadly thermophilic organism.

BACKGROUND: Primary amebic meningoencephalitis (PAM), caused by the free-living ameba Naegleria fowleri, has historically been associated with warm freshwater exposures at lower latitudes of the United States. In August 2010, a Minnesota resident, aged 7 years, died of rapidly progressive meningoencephalitis after local freshwater exposures, with no history of travel outside the state. PAM was suspected on the basis of amebae observed in cerebrospinal fluid. METHODS: Water and sediment samples were collected at locations where the patient swam during the 2 weeks preceding illness onset. Patient and environmental samples were tested for N. fowleri with use of culture and real-time polymerase chain reaction (PCR); isolates were genotyped. Historic local ambient temperature data were obtained. RESULTS: N. fowleri isolated from a specimen of the patient's brain and from water and sediment samples was confirmed using PCR as N. fowleri genotype 3. Surface water temperatures at the times of collection of the positive environmental samples ranged from 22.1°C to 24.5°C. August 2010 average air temperature near the exposure site was 25°C, 3.6°C above normal and the third warmest for August in the Minneapolis area since 1891. CONCLUSIONS: This first reported case of PAM acquired in Minnesota occurred 550 miles north of the previously reported northernmost case in the Americas. Clinicians should be aware that N. fowleri-associated PAM can occur in areas at much higher latitude than previously described. Local weather patterns and long-term climate change could impact the frequency of PAM.

Demostración del ameboflagelado Naegleria fowleri como agente etiológico de meningoencefalitis amébica primaria en Santiago de Cuba / Demonstration of flagellum ameba Naegleria fowleri as etiological agent of primary amebic meningoencephalitis in Santiago de Cuba

En los últimos años se ha demostrado que el amebo-flagelado Naegleria fowleri es el causante de la meningoencefalitis amébica primaria (MEAP). En Cuba ha sido mencionada la sospecha de su presencia en un líquido céfalo-raquídeo (LCR) de una ameba cuya locomoción y circunstancias sugería que pudiera ser esta ameba. Se realizó el análisis en fresco y en cultivo, con Test de Flagelación de 64 muestras de LCR seleccionadas al azar a partir de las 173 que presentaron las características de transparencia y negatividad a bacterias, requisitos iniciales en búsqueda de este agente, partiendo de una muestra inicial de 1 488 muestras de LCR procedentes de pacientes con meningoencefalitis clínicamente establecidas. De estos 64 LCR, 2 resultaron positivos a la evaluación por los diferentes métodos lo que permitió teñirlos con Giemsa, demostrar su capacidad de flagelación y su resistencia a TMT. Las características citoquímicas de los líquidos concordaban con lo planteado en la literatura y uno de los pacientes sobrevivió, el otro falleció. La patogenicidad de los ameboflagelados aislados de esas dos muestras de LCR se comprobó en el modelo animal ratón albino suizo y fueron observados al microscopio electrónico. Lo anterior confirma microbiológicamente por primera vez en el país este agente etiológico, aunque ya había elementos clínicos y de laboratorio de sospecha en casos anteriores(AU)

During the last years it has been demonstrated that the flagellae-carring ameba Naegleria fowleri is the aethiologic agent of the primary amoebic meningoencephalitis (PAME), in Cuba the suspicion of its presence has been mentioned in a Cefalo Raquideal Fluid (CRF) of an ameba whose locomotion and circumstances suggested that it could be this organism. The present work carries out the analysis in fresh CRF samples and after culture, performing the Flagelation Test of the 64 CRF that presented the characteristics of transparency and negativity to bacterial culture ,needed to suspicion of this flagellae-carring ameba, leaving of an starting sample of 1488 CRF.From this analysed sample of CRF only two were positive to the observation of amebas what allowed to tint them with Giemsa and also with Lugol, to demonstrate their flagelation capacity and their resistance to TMT in the corresponding test. The citochemical characteristics of the liquids agreed with that outlined in the literature for this organisms,and one of the patients survived, the other one died. The patogenicity of the isolated flagellae-carring amebas was proven in the animal model of Swiss albino mouse and both isolated strains were observed by the electronic microscope. This results are the basis for considering the present report the first one confirming N.Fowleri as aetiologic agent in two cases of PAME in Cuba(AU)

Demostración del ameboflagelado Naegleria fowleri como agente etiológico de meningoencefalitis amébica primaria en Santiago de Cuba / Demonstration of flagellum ameba Naegleria fowleri as etiological agent of primary amebic meningoencephalitis in Santiago de Cuba

En los últimos años se ha demostrado que el amebo-flagelado Naegleria fowleri es el causante de la meningoencefalitis amébica primaria (MEAP). En Cuba ha sido mencionada la sospecha de su presencia en un líquido céfalo-raquídeo (LCR) de una ameba cuya locomoción y circunstancias sugería que pudiera ser esta ameba. Se realizó el análisis en fresco y en cultivo, con Test de Flagelación de 64 muestras de LCR seleccionadas al azar a partir de las 173 que presentaron las características de transparencia y negatividad a bacterias, requisitos iniciales en búsqueda de este agente, partiendo de una muestra inicial de 1 488 muestras de LCR procedentes de pacientes con meningoencefalitis clínicamente establecidas. De estos 64 LCR, 2 resultaron positivos a la evaluación por los diferentes métodos lo que permitió teñirlos con Giemsa, demostrar su capacidad de flagelación y su resistencia a TMT. Las características citoquímicas de los líquidos concordaban con lo planteado en la literatura y uno de los pacientes sobrevivió, el otro falleció. La patogenicidad de los ameboflagelados aislados de esas dos muestras de LCR se comprobó en el modelo animal ratón albino suizo y fueron observados al microscopio electrónico. Lo anterior confirma microbiológicamente por primera vez en el país este agente etiológico, aunque ya había elementos clínicos y de laboratorio de sospecha en casos anteriores

During the last years it has been demonstrated that the flagellae-carring ameba Naegleria fowleri is the aethiologic agent of the primary amoebic meningoencephalitis (PAME), in Cuba the suspicion of its presence has been mentioned in a Cefalo Raquideal Fluid (CRF) of an ameba whose locomotion and circumstances suggested that it could be this organism. The present work carries out the analysis in fresh CRF samples and after culture, performing the Flagelation Test of the 64 CRF that presented the characteristics of transparency and negativity to bacterial culture ,needed to suspicion of this flagellae-carring ameba, leaving of an starting sample of 1488 CRF.From this analysed sample of CRF only two were positive to the observation of amebas what allowed to tint them with Giemsa and also with Lugol, to demonstrate their flagelation capacity and their resistance to TMT in the corresponding test. The citochemical characteristics of the liquids agreed with that outlined in the literature for this organisms,and one of the patients survived, the other one died. The patogenicity of the isolated flagellae-carring amebas was proven in the animal model of Swiss albino mouse and both isolated strains were observed by the electronic microscope. This results are the basis for considering the present report the first one confirming N.Fowleri as aetiologic agent in two cases of PAME in Cuba

Unexpected postmortem diagnosis of acanthamoeba meningoencephalitis in an immunocompetent child.

Meningoencephalitis caused by Acanthamoeba spp. is a rare opportunistic infection, difficult to diagnose and treat, which causes death in almost all cases. Here, the authors report a 5-year-old Iranian immunocompetent girl who died of fulminant acanthamoeba meningoencephalitis. To the authors' knowledge, this is the first case of acanthamoeba meningoencephalitis in Iran.

First case of granulomatous amebic encephalitis caused by Acanthamoeba castellanii in Taiwan.

We report the first case of granulomatous amebic encephalitis caused by Acanthamoeba in a previously healthy farmer in Taiwan who fell into a ditch. The DNA amplicons of amebas identified by polymerase chain reaction in the cerebrospinal fluid specimen and the ditch water specimens were identical and shared strong homology with A. castellanii . He survived after treatment with amphotericin B, rifampin, and corticosteroids.

Unexpected postmortem diagnosis of Acanthamoeba meningoencephalitis following allogeneic peripheral blood stem cell transplantation.

Meningoencephalitis caused by pathogenic free-living amebas is usually fatal. Only a few cases of Acanthamoeba meningoencephalitis, diagnosed at autopsy, have been reported following hematopoietic stem cell transplantation. We here report a case of Acanthamoeba meningoencephalitis following allogeneic peripheral blood stem cell transplantation with rapidly evolving neurologic symptoms that remained unexplained. Magnetic resonance imaging failed to show brain lesions and cerebrospinal fluid was negative for microbiological cultures. Definite diagnosis was an unexpected autopsy finding. As overall and teaching hospital autopsy rates are declining worldwide, we must emphasize the need of autopsy exams if we want to improve our knowledge as the best way to care for our patients.

Early diagnosis of Acanthamoeba infection during routine cytological examination of cerebrospinal fluid.

Early identification of Acanthamoeba in cerebrospinal fluid is mandatory to prevent fatal granulomatous amebic encephalitis. In the case presented here amebic trophozoites were detected in a routine cerebrospinal fluid sample. The antibiotic treatment and the apparently low virulence of this isolate were responsible for the benign progression of the infection.

Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I.

In this work, we analyzed the in vitro interaction of human secretory immunoglobulin A (sIgA) antibodies with Naegleria fowleri trophozoites and the capacity of these antibodies to inhibit amoeba adherence to collagen type I. We also studied N. fowleri antigens that are recognized by sIgA, using immunoblot assays. Immunocytochemical analysis of the interaction showed a redistribution of antigens on the surface of trophozoites by sIgA antibodies. Ultrastructural analysis of antibody-amoeba interaction showed that besides the patching and cap formation, parasites were capable of eliminating the antigen-antibody complex produced on the surface. sIgA antibodies were capable of inhibiting the in vitro adhesion of trophozoites to collagen type I. We suggest that nonsymptomatic infections by N. fowleri may stimulate a local specific immunity that prevents trophozoite adhesion and invasion of nasal mucosa.

Naegleria meningitis: a rare survival.

Acute amebic meningoencephalitis caused by free-living amebae naegleria fowleri is extremely rare and uniformly fatal with only seven survivals reported till date. An interesting case of naegleria meningitis diagnosed by wet mount cytology of cerebrospinal fluid (CSF) and treated with amphoterecin B, rifampicin and ornidazole with complete recovery is presented. In cases of suspected pyogenic meningitis, if CSF staining, antigen detection or culture is negative for bacteria, a wet mount cytology of CSF for naegleria is suggested. Early treatment with amphoterecin B and rifampicin may improve survival.

Primary amebic meningoencephalitis due to Naegleria fowleri: an autopsy case in Japan.

Free-living amebas represented by Naegleria fowleri, Acanthamoeba and Balamutia have been known to cause fatal meningoencephalitis since Fowler and Carter (1965) reported the first four human cases. An autopsy case of a 25-year-old female with primary amebic meningoencephalitis (PAM) due to Naegleria fowleri is described. Headache, lethargy and coma developed in this patient, and her condition progressed to death 8 days after the onset of clinical symptoms. Cerebral spinal fluid examination confirmed clusters of amebas, which were grown in culture and identified as Naegleria fowleri. At autopsy, lesions were seen in the central nervous system (CNS) and the ethmoid sinus. The CNS had severe, suppurative meningoencephalitis with amebic trophozoites mingled with macrophages. This case is the first report of PAM due to Naegleria fowleri in Japan.