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.

Balamuthia mandrillaris: the multiple nuclei of Balamuthia amebas; their location, activity, and site of development.

Multiple nuclei were first noted in the pseudopodia of Balamuthia mandrillaris amebas feeding on mammalian cells. Phase microscope observations of live amebas in vitro reveal that while many amebas have a single nucleus, others have multiple nuclear-like structures, now confirmed as nuclei with hematoxylin and Feulgen stains. In the live cultures, two nuclei located near the tip of an extended pseudopodium were seen to fuse resulting in one larger morphologic unit. Such merging of nuclei has not been previously reported. Other nuclei were located at positions that subsequently became the site for the outgrowth of an additional pseudopod branch. A newly discovered large structure, a polyploid nucleus, was located in the mid-part of the ameba. Nucleoli of uniform size were seen to develop from the central mass of chromatin and each became surrounded by a vesicular component as they moved into the protoplasm as morphologically complete nuclei.

The ameba Balamuthia mandrillaris feeds by entering into mammalian cells in culture.

Microscopic observations of live cultures of the pathogenic ameba Balamuthia mandrillaris and mammalian cells showed that amebic feeding involved the invasion of the pseudopodia, and/or the whole ameba into the cells. The ameba, recognized by their size and flow of organelles in the cytosol, was seen to extend the tip of a pseudopodium into the cytoplasm of a cell where it moved about leaving visible damage when retracted. In rounded cells, whole amebas were seen to enter into and move around before exiting a cell and then remain quiescent for hours. The invaded mammalian cells retained their turgidity and excluded vital dyes until only their denuded nuclei remained. The cytoplasm of the cells was consumed first, then the nuclei, but not their mitotic chromosomes. The feeding pattern of four isolates of B. mandrillaris, two from humans and two from soil samples, was by amebic invasion into the mammalian cells. The resulting ameba population included cysts, amebas on the surface, and free-floating amebas as individuals or in dense-packed clusters. There was no morphologic indication of a cytopathic change in the mammalian cells before their invasion by the amebas. Feeding by cell invasion is a distinctive feature of B. mandrillaris.

Development, characterization, and epitope mapping of a panel of twenty-four monoclonal antibodies specific for human inducible nitric oxide synthase.

A panel of monoclonal antibodies (MAbs) to human inducible nitric oxide synthase (hiNOS) has been developed. By isotype analysis of the MAbs cloned from the 24 different positive hybridomas, 13 were determined to be mouse IgG1, two were mouse IgG2a, two were mouse IgG2b, and the seven others were mouse IgM antibodies: all contained kappa light chains. The anti-hiNOS MAbs were initially characterized by ELISA, RIA, Western blot, and immunocytochemistry, and then they were epitope mapped using synthetic peptides and a three-step mapping procedure. In the first step, each of the 24 MAbs was tested by indirect ELISA for binding to 96 overlapping 18-amino acid-long peptides that span the entire 1153-amino acid length of hiNOS. Eight IgG class anti-hiNOS MAbs were found to bind to one of five different peptides. In the second step, a series of amino terminal and carboxyl terminal truncated peptides were synthesized for each of the five peptides to which one or more of the MAbs bound. Each of the eight anti-hiNOS MAbs was found to bind to the truncated peptides with a unique specificity that identified the amino acid segment involved in binding. The third step in the epitope mapping process utilized three series of overlapping 5-, 6-, 7-, 8-, and 9-amino acid-long peptides for each of these segments and identified the exact amino acids of hiNOS involved in antibody binding. Anti-hiNOS MAbs 2A1-F8, 2D2-B2, 21C10-1D10, and 24B10-2C7 were found to be especially useful in different immunoassays.

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.

Environmental isolation of Balamuthia mandrillaris associated with a case of amebic encephalitis.

This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmental soil sample associated with a fatal case of amebic encephalitis in a northern California child. Isolation of the ameba into culture from autopsied brain tissue confirmed the presence of Balamuthia: In trying to locate a possible source of infection, soil and water samples from the child's home and play areas were examined for the presence of Balamuthia: The environmental samples (plated onto nonnutrient agar with Escherichia coli as a food source) contained, in addition to the ameba, a variety of soil organisms, including other amebas, ciliates, fungi, and nematodes, as contaminants. Presumptive Balamuthia amebas were recognized only after cultures had been kept for several weeks, after they had burrowed into the agar. These were transferred through a succession of nonnutrient agar plates to eliminate fungal and other contaminants. In subsequent transfers, axenic Naegleria amebas and, later, tissue cultures (monkey kidney cells) served as the food source. Finally, the amebas were transferred to cell-free axenic medium. In vitro, the Balamuthia isolate is a slow-growing organism with a generation time of approximately 30 h and produces populations of approximately 2 x 10(5) amebas per ml. It was confirmed as Balamuthia by indirect immunofluorescence staining with rabbit anti-Balamuthia serum and human anti-Balamuthia antibody-containing serum from the amebic encephalitis patient. The environmental isolate is similar in its antimicrobial sensitivities and identical in its 16S ribosomal DNA sequences to the Balamuthia isolate from the deceased patient.