Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30 °C, 37 °C, and 40 °C) and osmotolerance (0.5 M, 1 M, and 1.5 M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Monocytes maintain central nervous system homeostasis following helminth-induced inflammation.

Neuroimmune interactions are crucial for regulating immunity and inflammation. Recent studies have revealed that the central nervous system (CNS) senses peripheral inflammation and responds by releasing molecules that limit immune cell activation, thereby promoting tolerance and tissue integrity. However, the extent to which this is a bidirectional process, and whether peripheral immune cells also promote tolerance mechanisms in the CNS remains poorly defined. Here we report that helminth-induced type 2 inflammation promotes monocyte responses in the brain that are required to inhibit excessive microglial activation and host death. Mechanistically, infection-induced monocytes express YM1 that is sufficient to inhibit tumor necrosis factor production from activated microglia. Importantly, neuroprotective monocytes persist in the brain, and infected mice are protected from subsequent lipopolysaccharide-induced neuroinflammation months after infection-induced inflammation has resolved. These studies demonstrate that infiltrating monocytes promote CNS homeostasis in response to inflammation in the periphery and demonstrate that a peripheral infection can alter the immunologic landscape of the host brain.

Heme oxygenase-1 modulates brain inflammation and apoptosis in mice with angiostrongyliasis.

The rat nematode lungworm Angiostrongylus cantonensis undergoes obligatory intracerebral migration in its hosts and causes eosinophilic meningitis or meningoencephalitis. Heme oxygenase 1 (HO-1) has several cytoprotective properties such as anti-oxidative, anti-inflammatory, and anti-apoptotic effects. HO-1 in brain tissues was induced in A. cantonensis-infected group and showed positive modulation in cobalt protoporphyrin (CoPP)-treated groups. Assay methods for the therapeutic effect include western blot analysis, enzyme-linked immunosorbent assay, gelatin zymography, blood-brain barrier permeability evaluation and eosinophil count in cerebrospinal fluid. The combination of albendazole (ABZ) and CoPP significantly decreased pro-inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-5, and IL-33 but significantly increased anti-inflammatory cytokines IL-10 and transforming growth factor-ß. In addition, worm recovery, matrix metalloproteinase-9, BBB permeability, and eosinophil counts were decreased in the ABZ and CoPP co-treated groups. Induction of HO-1 with CoPP strongly inhibited the protein levels of caspase-3 and increased the induction of annexin-V and B-cell leukemia 2. Thus, co-treatment with ABZ and CoPP prevented A. cantonensis-induced eosinophilic meningoencephalitis and its anti-apoptotic effect by promoting HO-1 signaling prior to BBB dysfunction. HO-1 induction might be a therapeutic modality for eosinophilic meningoencephalitis.

Antimicrobial effect of auranofin against Acanthamoeba spp.

Acanthamoebae are opportunistic pathogens that cause serious infections, including Acanthamoeba keratitis, a sight-threatening disease affecting mainly contact lens wearers, and granulomatous amoebic encephalitis, an infection of the central nervous system that occurs mostly in immunocompromised individuals. Although these infections are rare, they are a challenge for healthcare providers. In the last decade, the search for and implementation of novel treatment approaches against these parasites and the infections they cause have intensified, but current options are still unsatisfactory. The aim of this study was to investigate the in vitro activity of the gold-based compound auranofin against Acanthamoeba spp. The study showed that auranofin has potent antimicrobial activity against Acanthamoeba spp., with an IC50 ranging from 2.9 to 3.48 µM, and thus may be useful in the prevention and control of Acanthamoeba infections.

Case Report: Granulomatous Amebic Encephalitis due to Acanthamoeba spp. in an Immunocompetent Pediatric Patient.

Granulomatous amebic encephalitis (GAE) caused by Acanthamoeba is a rare infection with central nervous system (CNS) involvement usually with fatal consequences. Currently, information regarding GAE in children is scarce and is limited only to case reports and case series. A 13-year-old immunocompetent male patient with a 6-month history of progressive and intermittent headaches presented to our institution. One week before hospital admission, the patient showed signs of CNS involvement. Magnetic resonance imaging revealed multiple lesions with supra- and infratentorial cerebral abscesses. An empiric treatment with combined antibiotics was given, but the patient died after 20 days of hospital stay. A postmortem diagnosis confirmed GAE. Although it is a rare disease in pediatric patients, GAE should be considered in children with a chronic history of fever, headache, and vomiting with CNS involvement.

Balamuthia mandrillaris encephalitis in a child: case report and literature review.

Balamuthia mandrillaris encephalitis is a rare disease with high mortality in the children. Due to the lack of specificity in clinical manifestations, laboratory tests, and neuroimaging, the diagnosis of the disease is difficult, especially the diagnosis of etiology. Currently, the evidence shows that the diagnosis of the disease depends on local brain biopsy or autopsy, and it is difficult to detect the pathogens by traditional etiological detection methods in blood and cerebrospinal fluid. We report a 9-year-old Chinese girl with B. mandrillaris encephalitis who was diagnosed with metagenomic next-generation sequencing (mNGS). The technology of mNGS can provide rapid, early etiological diagnosis without the need for a local brain biopsy, which can buy time for the early treatment of patients. We also provide a comprehensive literature review on this disease.

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles.

BACKGROUND: Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment. METHOLOGY/PRINCIPLE FINDINGS: Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-Isopropylacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. CONCLUSION/SIGNIFICANCES: Our results demonstrate nanoparticle technology's potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

Preventing contamination of PCR-based multiplex assays including the use of a dedicated biosafety cabinet.

We retrospectively investigated cases of false-positive diagnoses using the BIOFIRE® FilmArray® meningitis/encephalitis (ME) panel to measure the impact of using a dedicated biosafety cabinet combined with preventive measures to reduce the prevalence of false-positive diagnoses due to pre-analytical in-laboratory contamination. False-positive results were identified by reviewing clinical data, biological parameters and cytology results of cerebrospinal fluid (CSF) samples showing discrepant results between the FilmArray ME panel and routine PCR assays. A total of 327 CSF were analysed over 16 weeks in point-of-care (POC) A and B, over two 8-week periods, periods 1 and 2. The analysis yielded 30 (9·17%) detection of at least one pathogen including 21/30 (70%) viruses and 9/30 (30%) bacteria. During period 1, POC-A and POC-B manipulated CSF under a non-dedicated hood featuring laminar flow, whereas during period 2, CSFs were manipulated under a dedicated biosafety cabinet without any airflow in POC-A. During period 1, false positives were detected in 3/114 CSF (2·63%) in POC-A and 1/36 (2·77%) in POC-B (P = 0·97); during period 2, false positives were detected in 0/139 CSF (0%) in POC-A and 1/38 (2·63%) in POC-B (P = 0·23). All false positives were bacterial. The use of a dedicated cabinet without ventilation along with preventive measures during period 2 in POC-A significantly reduced the number of false-positive results (P = 0·05). Preventive measures described in this study can mitigate false positives when using PCR-based multiplex assays such as the BIOFIRE FilmArray ME Panel for the diagnosis of meningitis and other infectious diseases.

Diagnosing Balamuthia mandrillaris encephalitis via next-generation sequencing in a 13-year-old girl.

Balamuthia amoebic encephalitis has a subacute-to-chronic course and is almost invariably fatal owing to delayed diagnosis and a lack of effective therapy. Here, we report a 13-year-old girl with cutaneous lesions and multifocal granulomatous encephalitis. The patient underwent a series of tests and was suspected as having tuberculosis. She was treated with various empiric therapies without improvement. She was finally correctly diagnosed via next-generation sequencing of the cerebrospinal fluid. The patient deteriorated rapidly and died 2 months after being diagnosed with Balamuthia mandrillaris encephalitis. This study highlights the important clinical significance of next-generation sequencing, which provides better diagnostic testing for unexplained paediatric encephalitis, especially that caused by rare or emerging pathogens.

"Proposals for Amendments in the Diagnosis and Treatment of Encephalitis caused by Free-living Amoebae".

Encephalitis caused by Free-living amoebae (FLA) has a mortality rate of around 95- 98%, a fraction that has not changed in the past decades. Pathogenic FLA include Acanthamoeba, Balamuthia mandrillaris, and Naegleria fowleri that are known to target the brain after an extra cerebral infection in the case of Acanthamoeba and Balamuthia mandrillaris, or directly the brain, as in the case of the Naegleria fowleri. The Acanthamoeba spp. and Balamuthia mandrillaris cause granulomatous amoebic encephalitis (GAE) while Naegleria fowleri, the so termed "brain eating amoeba" causes primary amoebic meningoencephalitis (PAM). The attempts to obtain a speedy diagnosis and an aggressive treatment protocol are the areas where advances can make a difference and reduce the mortality rates. At first, we highlight the reasons behind the diagnostic delays and treatment failures and provide proposals to establish a quick diagnosis in both PAM and GAE. Secondly, we emphasize the use of a transcribrial device, and a prompt, but vigilant surgical reduction of the intracranial pressure in these patients which could be life-saving. We also debate that an exudate obtained from the olfactory region by irrigation via a modified transcribrial device or by conventional methods, instead of a cerebrospinal fluid sample, could serve as a source of obtaining amoeba in PAM for a real-time polymerase chain reaction-based definitive diagnosis of PAM. Also, introduced is the rationale that has the potential to deliver the drugs to the brain in patients with PAM and the GAE localized to the frontal lobe of the brain, by bypassing the blood brain barrier. We put forward these proposals for debate and deliberation to our fellow colleagues in order to spot the potential of their application to reduce the mortality rates caused by the rare but fatal encephalitis caused by these FLA.

Species composition, geographical distribution and seasonal abundance of the Anopheles maculipennis complex along the Upper Rhine, Germany.

Between May and September 2016, mosquitoes were collected on a biweekly basis at 55 locations with CO2-baited encephalitis vector surveillance traps along the Upper Rhine, Germany, to evaluate the species composition, geographical distribution and abundance of the Anopheles maculipennis complex, some members of this complex being considered vectors of historical malaria in Germany. A total of 2115 Anopheles maculipennis complex specimens were collected during the season, of which a sample of 1252 individuals was determined to species level by amplification of species-specific internal transcribed spacer 2 (ITS2) sequences. A total of 856 individuals of Anopheles daciae (68.37%), 394 Anopheles messeae (31.47%) and 2 Anopheles maculipennis s.s. (0.16%) were recorded. The number and proportion of A. daciae was remarkably higher in the northern meandering zone of the Upper Rhine (843 specimens, 79.90%), than in the more canalised southern furcation zone where A. messeae with 183 collected specimens represented 92.89% of 197 classified individuals. The average number of collected A. maculipennis s.l. individuals per trapping site was 38.45, equalling 0.64% of the total mosquito collection. Despite an increase in imported malaria cases, this comparatively low abundance of A. maculipennis s.l. may indicate a low risk of endemic malaria transmission by members of the A. maculipennis complex today. The proportionally dominance of A. daciae suggests that this species could be suspected the main historical vector of malaria in the Upper Rhine region. Sequence analyses of the ITS2 fragment revealed intraindividual polymorphisms within 3 of 5 diagnostic nucleotides in all specimens of A. daciae, raising the question if additional loci should be considered, to gain further insight into the taxonomical relation to A. messeae.

Pathogenic free-living amoebic encephalitis in Japan.

Over 600 cases of amoebic encephalitis caused by pathogenic free-living amoebas (Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri) have been reported worldwide, and in Japan, 24 cases have been reported from the first case in 1976 up to 2018. Among these cases, 18 were caused by B. mandrillaris, four by Acanthamoeba spp., one by N. fowleri, and one was of unknown etiology. Additionally, eight cases were diagnosed with encephalitis due to pathogenic free-living amoebas before death, but only three cases were successfully treated. Unfortunately, all other cases were diagnosed by autopsy. These facts indicate that an adequate diagnosis is difficult, because encephalitis due to pathogenic free-living amoebas does not show typical symptoms or laboratory findings. Moreover, because the number of cases is limited, other cases might have been missed outside of those diagnosed by autopsy. Cases of encephalitis caused by B. mandrillaris have been reported from all over Japan, with B. mandrillaris recently isolated from soil in Aomori prefecture. Therefore, encephalitis caused by pathogenic free-living amoebas should be added to the differential diagnosis of encephalitis patients.

Synthesis and in vitro activity of new biguanide-containing dendrimers on pathogenic isolates of Acanthamoeba polyphaga and Acanthamoeba griffini.

The genus Acanthamoeba can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the existence of a resistance stage that many times causes infection relapses. In an attempt to add new agents to our chemotherapeutic arsenal against acanthamebiasis, two Acanthamoeba isolates were treated in vitro with newly synthesized biguanide dendrimers. Trophozoite viability analysis and ultrastructural studies showed that dendrimers prevent encystment by lysing the cellular membrane of the amoeba. Moreover, one of the dendrimers showed low toxicity when tested on mammalian cell cultures, which suggest that it might be eventually used as an amoebicidal drug or as a disinfection compound in contact lens solutions.

MRI of Iron Oxide Nanoparticles and Myeloperoxidase Activity Links Inflammation to Brain Edema in Experimental Cerebral Malaria.

Purpose To investigate the association of inflammation and brain edema in a cerebral malaria (CM) mouse model with a combination of bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium, referred to as MPO-Gd, and cross-linked iron oxide nanoparticle (CLIO-NP) imaging. Materials and Methods Female wild-type (n = 23) and myeloperoxidase (MPO) knock-out (n = 5) mice were infected with the Plasmodium berghei ANKA strain from May 2016 to July 2018. Seven healthy mice served as control animals. At a Rapid Murine Coma and Behavioral Scale (RMCBS) score of less than 15, mice underwent MRI at 9.4 T and received gadodiamide, MPO-Gd, or CLIO-NPs. T1-weighted MRI was used to assess MPO activity, and T2*-weighted MRI was used to track CLIO-NPs. Immunofluorescent staining and flow cytometric analyses characterized CLIO-NPs, MPO, endothelial cells, and leukocytes. An unpaired, two-tailed Student t test was used to compare groups; Spearman correlation analysis was used to determine the relationship of imaging parameters to clinical severity. Results MPO-Gd enhancement occurred in inflammatory CM hotspots (olfactory bulb > rostral migratory stream > brainstem > cortex, P < .05 for all regions compared with control mice; mean olfactory bulb signal intensity ratio: 1.40 ± 0.07 vs 0.96 ± 0.01, P < .01). The enhancement was reduced in MPO knockout mice (mean signal intensity ratio at 60 minutes: 1.13 ± 0.04 vs 1.40 ± 0.07 in CM, P < .05). Blood-brain barrier compromise was suggested by parenchymal gadolinium enhancement, leukocyte recruitment, and endothelial activation. CLIO-NPs accumulated mainly intravascularly and at the vascular endothelium. CLIO-NPs were also found in the choroid plexus, indicating inflammation of the ventricular system. Blood-cerebrospinal fluid barrier breakdown showed correlation with brain swelling (r2: 0.55, P < .01) and RMCBS score (r2: 0.75, P < .001). Conclusion Iron oxide nanoparticle imaging showed strong inflammatory involvement of the microvasculature in a murine model of cerebral malaria. Furthermore, bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium imaging depicted parenchymal and intraventricular inflammation. This combined molecular imaging approach links vascular inflammation to breakdown of the blood-brain barrier and blood-cerebrospinal fluid barrier that correlate with global brain edema and disease severity. © RSNA, 2018 Online supplemental material is available for this article. See also the editorial by Kiessling in this issue.

Mice as paratenic hosts of Aelurostrongylus abstrusus.

BACKGROUND: Several species of nematodes included in the superfamily Metastrongyloidea are recognized agents of parasitic infections in felines. Aelurostrongylus abstrusus is the most prevalent species affecting the respiratory system of domestic cats. The route of infection in cats is supposed to be through ingestion of gastropod intermediate or paratenic hosts. However, because gastropods are not the preferred preys of cats, rodents were suggested to play an important role as paratenic hosts in the biological cycle of A. abstrusus and in the epidemiology of aelurostrongylosis. RESULTS: Two studies were conducted to document histopathological tissue lesions in mice experimentally infected with A. abstrusus third-stage larvae (L3) (Study 1), and to determine larval counts in their organs (Study 2). Additionally, cats were fed with experimentally infected mice to assess their infectivity. Aelurostrongylus abstrusus L3 were recovered from the liver, spleen, brain, skeletal muscle and gastrointestinal tract tissues by artificial digestion, and heart, spleen and brain tested positive for A. abstrusus at molecular diagnosis. Multifocal encephalitis and meningitis and glial nodules were the most common histopathological lesions found in mice inoculated with A. abstrusus. All cats shed first-stage larvae of A. abstrusus after ingestion of mice inoculated with this nematode. CONCLUSIONS: In this study, we provide information on the anatomical localization, histopathological alterations and rate of recovery of A. abstrusus L3 in mice, and confirm their infectivity to cats (definitive hosts) after feeding on infected mice (paratenic hosts). Data presented here add knowledge to further understand the biology of A. abstrusus in mice and underline the importance of mice as paratenic hosts of this nematode for the infection of cats.

Clinical Features, Diagnosis, and Outcome of Encephalitis in French Guiana.

The aim of our study was to describe the clinical features, the etiologies, and the factors associated with poor outcome of encephalitis in French Guiana. Our study was retrospective, including all cases of encephalitis hospitalized in the Cayenne General Hospital, from January 2007 to July 2017. Patients were included through the 2013 encephalitis consortium criteria and the outcome was evaluated using the Glasgow outcome scale at 3 months from the diagnosis of encephalitis. We included 108 patients, giving an approximate incidence rate of four cases/100,000 inhabitants/year. The origin of the encephalitis was diagnosed in 81 cases (75%), and 72 of them (66.7%) were from an infectious origin. The most common infectious causes were Cryptococcus sp. (18.5%) independently of the immune status, Toxoplasma gondii (13.9%), and Streptococcus pneumoniae (5.5%). In the follow-up, 48 patients (46.6%) had poor outcome. Independent risk factors associated with poor outcome at 3 months were "coming from inside area of the region" (P = 0.036, odds ratio [OR] = 4.19; CI 95% = 1.09-16.06), need for mechanical ventilation (P = 0.002, OR = 5.92; CI 95% = 1.95-17.95), and age ≥ 65 years (P = 0.049, OR = 3.99; CI 95% = 1.01-15.89). The most identified cause of encephalitis in French Guiana was Cryptococcus. The shape of the local epidemiology highlights the original infectious situation with some local specific pathogens.

Astrogliopathology in the infectious insults of the brain.

Astroglia, a heterogeneous type of neuroglia, play key homeostatic functions in the central nervous system (CNS) and represent an important defence system. Impaired homeostatic capacity of astrocytes manifests in diseases and this is mirrored in various astrocyte-based pathological features including reactive astrogliosis, astrodegeneration with astroglial atrophy and pathological remodelling of astrocytes. All of these manifestations are most prominently associated with infectious insults, mediated by bacteria, protozoa and viruses. Here we focus onto neurotropic viruses such as tick-borne encephalitis (TBEV) and Zika virus (ZIKV), both belonging to Flaviviridae and both causing severe neurological impairments. We argue that astrocytes provide a route through which neurotropic infectious agents attack the CNS, since they are anatomically associated with the blood-brain barrier and exhibit aerobic glycolysis, a metabolic specialisation of highly morphologically dynamic cells, which may provide a suitable metabolic milieu for proliferation of infectious agents, including viral bodies.

Metagenomic next-generation sequencing as a diagnostic tool for toxoplasmic encephalitis.

BACKGROUND: More than 100 different pathogens can cause encephalitis. Testing of all the neurological pathogens by conventional methods can be difficult. Metagenomic next-generation sequencing (NGS) could identify the infectious agents in a target-independent manner. The role of this novel method in clinical diagnostic microbiology still needs to be evaluated. In present study, we used metagenomic NGS to search for an infectious etiology in a human immunodeficiency virus (HIV)-infected patient with lethally diffuse brain lesions. Sequences mapping to Toxoplasma gondii were unexpectedly detected. CASE PRESENTATION: A 31-year-old HIV-infected patient presented to hospital in a critical ill condition with a Glasgow coma scale score of 3. Brain magnetic resonance imaging showed diffuse brain abnormalities with contrast enhancement. Metagenomic NGS was performed on DNA extract from 300 µL patient's cerebrospinal fluid (CSF) with the BGISEQ-50 platform. The sequencing detection identified 65,357 sequence reads uniquely aligned to the Toxoplasma gondii genome. Presence of Toxoplasma gondii genome in CSF was further verified by Toxoplasma gondii-specific polymerase chain reaction and Sanger sequencing. Altogether, those results confirmed the diagnosis of toxoplasmic encephalitis. CONCLUSIONS: This study suggests that metagenomic NGS may be a useful diagnostic tool for toxoplasmic encephalitis. As metagenomic NGS is able to identify all pathogens in a single run, it may be a promising strategy to explore the clinical causative pathogens in central nervous system infections with atypical features.

Non-granulomatous cerebellar infection by Acanthamoeba spp. in an immunocompetent host.

Acanthamoeba spp. is a free-living amoeba, frequently involved in keratitis by contact lens in immunocompetent hosts. Anecdotal reports associate Acanthamoeba spp. as a cause of severe granulomatous encephalitis in immunocompromised and, less frequently, in immunocompetent subjects. Data regarding clinical and therapeutic management are scanty and no defined therapeutic guidelines are available. We describe an unusual case of non-granulomatous Acanthamoeba cerebellitis in an immunocompetent adult male, with abrupt onset of neurological impairment, subtle hemorrhagic infarction at magnetic resonance imaging, and initial suspicion of cerebellar neoplasm. Histopathological findings of excised cerebellar mass revealed the presence of necrosis and inflammation with structure resembling amoebic trophozoites, but without granulomas. Polymerase chain reaction from cerebellar tissue was positive for Acanthamoeba T4 genotype. Due to gastrointestinal intolerance to miltefosine, the patient was treated with long-term course of fluconazole and trimethoprim/sulphamethoxazole, obtaining complete clinical and neuroradiological resolution.