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.

Identification and molecular typing of Naegleria fowleri from a patient with primary amebic meningoencephalitis in China.

Naegleria fowleri is the only Naegleria spp. known to cause an acute, fulminant, and rapidly fatal central nervous system infection in humans called primary amebic meningoencephalitis (PAM). In 2016, a patient with suspected PAM was found in Zhejiang Province of China. The pathogen was identified by microscopic examination and PCR. The positive PCR products were sequenced and the sequences were aligned using the NCBI BLAST program. The homologous and phylogenetic analysis was conducted using MEGA 6 program. On microscopy of direct smears, motile cells with pseudopodia were observed, and the motion characteristics of the pseudopodia as well as the cell morphology suggested that the pathogens were amoeba trophozoites. Wright-Giemsa-stained smears showed amoeba trophozoites of various shapes, which measured 10-25µm in size; these were characterized by a prominent, centrally placed nucleolus and a vacuolated cytoplasm. PCR was negative for Entamoeba histolytica and Entamoeba dispar, but positive for Naegleria spp. and N. fowleri. The nucleotide sequences acquired in this study have been submitted to GenBank with accession numbers KX909928 and KX909927, respectively. The BLAST analysis revealed that the sequences of KX909928 and KX909927 had 100% similarity with the sequence of the N. fowleri gene (KT375442.1). Sequence alignment and the phylogenetic tree revealed that the N. fowleri collected in this study was classified as genotype 2 and was most closely related to Naegleria lovaniensis. This study confirmed N. fowleri as the agent responsible for the infection in this patient. PAM normally progresses rapidly and is generally universally fatal within a week. Unfortunately this patient died at 2 weeks after the onset of symptoms.

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.

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.

Matrix metalloproteinase-9 and intercellular adhesion molecule 1 are powerful staging markers for human African trypanosomiasis.

OBJECTIVES: A critical step before treatment of human African trypanosomiasis (HAT) is the correct staging of the disease. As late stage is established when trypanosomes cross the blood-brain barrier and invade the central nervous system, we hypothesized that matrix metalloproteinases and cell adhesion molecules could indicate, alone or in combination, the disease progression from the first to the second stage of HAT. METHODS: We measured the levels of MMP-2, MMP-9, ICAM-1, VCAM-1 and E-selectin in the cerebrospinal fluid (CSF) of 63 Trypanosoma brucei gambiense-infected patients (15 stage 1 and 48 stage 2). Staging was based on counting of white blood cells (WBC) and/or parasite detection in CSF. Concentrations were obtained either by ELISA or multiplex bead suspension assays, and results were compared with three known HAT staging markers (CXCL10, CXCL8 and H-FABP). RESULTS: ICAM-1 and MMP-9 accurately discriminated between stage 1 and stage 2 patients with HAT with 95% sensitivity (SE) for 100% specificity (SP), which was better than CXCL10 (93% SE for 100% SP), one of the most promising known markers. Combination of ICAM-1 and MMP-9 with H-FABP provided a panel that resulted in 100% of SE and SP for staging HAT. CONCLUSIONS: ICAM-1 and MMP-9, alone or in combination, appeared as powerful CSF staging markers of HAT. Final validation of all newly discovered staging markers on a large multi-centric cohort including both forms of the disease as well as patients with others infections should be performed.

[Molecular diagnosis of a fatal primary amoebic meningoencephalitis in Guadeloupe (French West Indies)]. / Diagnostic moléculaire d'une méningoencéphalite amibienne primitive à l'occasion d'un cas fatal en Guadeloupe.

We report the first case of primary amoebic meningoencephalitis in a 9-year-old boy in Guadeloupe. The outcome was rapidly fatal in 7 days. The patient presumably acquired the infection by swimming and diving in a basin supplied by natural thermal water 1 week before onset of the disease. The possibility of a free-living amoeba infection was suspected both on the negativity of all bacterial and viral initial tests and on the observation of peculiar cells in stained cerebrospinal fluid samples. Although the amoeba was not isolated, Naegleria fowleri could be identified by polymerase chain reaction with specific primers on DNA extracted from frozen cerebrospinal fluid samples. Furthermore, as the internal transcribed spacer (ITS1) region of DNA is variable in length between the different strains of N. fowleri, sequencing of the amplified ITS1 demonstrated that the responsible N. fowleri strain belongs to a common genotype present in the American and European continent.

[Human African trypanosomiasis in mangrove epidemiologic area. Presentation, diagnosis and treatment in Guinea, 2005-2007]. / La trypanosomose humaine africaine en faciès épidémiologique de mangrove. Présentation, déterminants et prise en charge dans le contexte de la Guinée (2005 à 2007).

UNLABELLED: Gambiense human African trypanosomiasis is still assumed to be endemic in many part of West Africa, particularly in Guinea coastal area with mangrove swamp. Diagnosis is usually made during active medical screening or by passive initiative. OBJECTIVES: To describe clinical and epidemiological characteristics of Gambiense human African trypanosomiasis in the coastal area of Guinea. METHODS: Exhaustive and retrospective analysis of all patients attending the trypanosomiasis center in the coastal area of Guinea between January 2005 and December 2007 with a diagnosis of human African trypanosomiasis. RESULTS: A total of 196 patients were recruited for the study. Out of them, 55 % of the 73 patients diagnosed during active screening were classified stage 1 (haemolymphatic stage) or early stage 2 (meningoencephalitic stage). Contrarily, 115 of the 120 diagnosed by passive procedure were classified late stage 2, which features more specific signs and neurological symptoms, and leads to coma and death. More than 90 % of all cases presented cervical lymph nodes with identification of trypanosome on direct examination of fluid puncture. Less than one third of the patients were reexamined three months later. DISCUSSION: In the coastal area of Guinea with mangrove swamp, direct examination of lymph node fluid puncture seems to be the most contributive test for the diagnosis of human African trypanosomiasis. Hence, associating clinical examination of cervical lymph nodes area and direct examination of fluid puncture may allow an early diagnosis of Gambiense human African trypanosomiasis and favor the implementation of efficient therapeutic strategies.

Cytokines in central nervous system trypanosomiasis: cause, effect or both?

The late, or encephalitic, stage of human African trypanosomiasis (HAT), or sleeping sickness, is typified by a diffuse meningoencephalitis characterised neuropathologically by perivascular infiltration of inflammatory cells. While the cause of this neuroinflammatory reaction is not understood, there is evidence for the roles of pro-inflammatory cytokines such as IFN-gamma and TNF-alpha and counter-inflammatory cytokines such as IL-10, with the balance of these influencing disease outcome. Because of the practical difficulties of obtaining serial measurements in patients, it has proved difficult to assign either cause or effect properties to measured cytokines, but mechanistic animal modelling studies are proving helpful.

Diagnosing central nervous system trypanosomiasis: two stage or not to stage?

One of the most difficult issues in the management of patients with human African trypanosomiasis (HAT) is the reliable distinction between early-stage disease and late-stage disease where the trypanosomes have traversed the blood-brain barrier to enter the central nervous system (CNS). Currently, there is no universal consensus for the cerebrospinal fluid (CSF) findings that define late-stage HAT and provide the rationale for treatment with toxic drugs for CNS disease. Whilst some clinicians use the WHO CSF criteria, others use alternative findings to define late-stage disease. Novel analyses in CSF of patients are urgently required for more accurate diagnostic staging.

Comparative analysis of cytokine gene expression in cerebrospinal fluid of horses without neurologic signs or with selected neurologic disorders.

OBJECTIVE: To determine gene transcription for cytokines in nucleated cells in CSF of horses without neurologic signs or with cervical stenotic myelopathy (CSM), West Nile virus (WNV) encephalitis, equine protozoal myeloencephalitis (EPM), or spinal cord trauma. ANIMALS: 41 horses (no neurologic signs [n = 12], CSM [8], WNV encephalitis [9], EPM [6], and spinal cord trauma [6]). PROCEDURES: Total RNA was extracted from nucleated cells and converted into cDNA. Gene expression was measured by use of real-time PCR assay and final quantitation via the comparative threshold cycle method. RESULTS: Cytokine genes expressed by nucleated cells of horses without neurologic signs comprised a balance between proinflammatory tumor necrosis factor-alpha (TNF-alpha), anti-inflammatory cytokines (interleukin [IL]-10 and transforming growth factor [TGF]-beta), and Th1 mediators (interferon [IFN]-gamma). Cells of horses with CSM mainly expressed genes for TNF-alpha, TGF-beta, and IL-10. Cells of horses with WNV encephalitis mainly expressed genes for IL-6 and TGF-beta. Cells of horses with EPM mainly had expression of genes for IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, and TGF-beta. Cells from horses with spinal cord trauma had expression mainly for IL-6; IFN-gamma; TGF-beta; and less frequently, IL-2, IL-10, and TNF-alpha. Interleukin-8 gene expression was only detected in CSF of horses with infectious diseases. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the small number of CSF samples for each group, results suggest distinct gene signatures expressed by nucleated cells in the CSF of horses without neurologic signs versus horses with inflammatory or traumatic neurologic disorders.

Indirect fluorescent antibody testing of cerebrospinal fluid for diagnosis of equine protozoal myeloencephalitis.

OBJECTIVE: To assess the use of CSF testing with an indirect fluorescent antibody test (IFAT) for diagnosis of equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona. SAMPLE POPULATION: Test results of 428 serum and 355 CSF samples from 182 naturally exposed, experimentally infected, or vaccinated horses. PROCEDURE: EPM was diagnosed on the basis of histologic examination of the CNS. Probability distributions were fitted to serum IFAT results in the EPM+ and EPM-horses, and correlation between serum and CSF results was modeled. Pairs of serum-CSF titers were generated by simulation, and titer-specific likelihood ratios and post-test probabilities of EPM at various pretest probability values were estimated. Post-test probabilities were compared for use of a serum-CSF test combination, a serum test only, and a CSF test only. RESULTS: Post-test probabilities of EPM increased as IFAT serum and CSF titers increased. Post-test probability differences for use of a serum-CSF combination and a serum test only were < or = 19% in 95% of simulations. The largest increases occurred when serum titers were from 40 to 160 and pre-test probabilities were from 5% to 60%. In all simulations, the difference between pre- and post-test probabilities was greater for a CSF test only, compared with a serum test only. CONCLUSIONS AND CLINICAL RELEVANCE: CSF testing after a serum test has limited usefulness in the diagnosis of EPM. A CSF test alone might be used when CSF is required for other procedures. Ruling out other causes of neurologic disease reduces the necessity of additional EPM testing.

Cerebral trypanosomiasis and AIDS.

UNLABELLED: A 36 year-old black female, complaining of headache of one month's duration presented with nausea, vomiting, somnolence, short memory problems, loss of weight, and no fever history. Smoker, intravenous drugs abuser, promiscuous lifestyle. PHYSICAL EXAMINATION: left homonimous hemianopsia, left hemiparesis, no papilledema, diffuse hyperreflexia, slowness of movements. Brain CT scan: tumor-like lesion in the splenium of the corpus calosum, measuring 3.5 x 1.4 cm, with heterogeneous enhancing pattern, suggesting a primary CNS tumor. Due to the possibility of CNS infection, a lumbar puncture disclosed an opening pressure of 380 mmH(2)0; 11 white cells (lymphocytes); glucose 18 mg/dl (serum glucose 73 mg/dl); proteins 139 mg/dl; presence of Trypanosoma parasites. Serum Elisa-HIV tests turned out to be positive. Treatment with benznidazole dramatically improved clinical and radiographic picture, but the patient died 6 weeks later because of respiratory failure. T. cruzi infection of the CNS is a rare disease, but we have an increasing number of cases in HIV immunocompromised patients. Diagnosis by direct observation of CSF is uncommon, and most of the cases are diagnosed by pathological examination. It is a highly lethal disease, even when properly diagnosed and treated. This article intends to include cerebral trypanosomiasis in the differential diagnosis of intracranial space-occupying lesions, especially in immunocompromised patients from endemic regions.

Lymphocyte phenotype subsets in the cerebrospinal fluid of normal horses and horses with equine protozoal myeloencephalitis.

The percentages of T-lymphocytes, lymphocyte subsets CD4+ and CD8+ T-cells, and lymphocyte adhesion molecule CD11a/CD18 were determined in the peripheral blood and cerebrospinal fluid (CSF) of seven normal horses and four horses with equine protozoal myeloencephalitis (EPM) using flow cytometry. There was a greater percentage of CD5+ cells in the CSF (79.0%) than in peripheral blood (67.0%), although this did not achieve statistical significance. Furthermore, the lymphocyte population in CSF comprises a significantly greater (P = .01) percentage of CD8+ T-cells, resulting in a decrease of the CD4/CD8 ratio. Lymphocyte phenotype subsets in peripheral blood or CSF from horses affected with EPM did not differ from normal horses, although CD5+ T-lymphocytes were seen in significantly greater numbers in the CSF of EPM-affected horses (93.2%) than in normal horses (79.0%).