Bis-benzimidazole hits against Naegleria fowleri discovered with new high-throughput screens.

Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium- or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of <1 µM, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri.

Prompt diagnosis and extraordinary survival from Naegleria fowleri meningitis: a rare case report.

Primary amoebic meningoencephalitis is a rare fatal meningitis caused by free living amoeba Naegleria fowleri, found in freshwater ponds and lakes. It infects children and young adults with exposure due to swimming or diving. We report a case of N. fowleri meningitis in a 6-year-old boy who presented with signs and symptoms of acute bacterial meningitis. No history of travelling or swimming was present. However, the boy frequently played with water stored from a "kuhl" (diversion channels of water). Wet mount of cerebrospinal fluid (CSF) revealed amoeboid and actively motile flagellate forms of trophozoites. CSF culture done on 1.5% non-nutrient agar plates with a lawn culture of Escherichia coli kept at 37°C for 15 days did not reveal any growth. The test of flagellation on passing CSF in distilled water was however positive in 3 h. Water of the "kuhl" from the stored tank also showed actively motile trophozoites similar to the forms obtained from the CSF. Based on our reports, the boy was immediately treated with amphotericin B, rifampicin and fluconazole for 21 days. Repeat CSF examination after 14 days did not reveal any trophozoites in wet mount and patient was discharged after 3 weeks of successful treatment.

Survey of Naegleria fowleri in geothermal recreational waters of Guadeloupe (French West Indies).

In 2008 a fatal case of primary amoebic meningoencephalitis, due to the amoeboflagellate Naegleria fowleri, occurred in Guadeloupe, French West Indies, after a child swam in a bath fed with geothermal water. In order to improve the knowledge on free-living amoebae in this tropical part of France, we investigated on a monthly basis, the presence of Naegleria spp. in the recreational baths, and stream waters which feed them. A total of 73 water samples, 48 sediments and 54 swabs samples were collected from 6 sampling points between June 2011 and July 2012. The water samples were filtered and the filters transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli while sediment and swab samples were placed directly on these plates. The plates were incubated at 44°C for the selective isolation of thermophilic Naegleria. To identify the Naegleria isolates the internal transcribed spacers, including the 5.8S rDNA, were amplified by polymerase chain reaction and the sequence of the PCR products was determined. Thermophilic amoebae were present at nearly all collection sites. The pathogenic N. fowleri was the most frequently encountered thermophilic species followed by N. lovaniensis. The concentration of N. fowleri was rather low in most water samples, ranging from 0 to 22 per liter. Sequencing revealed that all N. fowleri isolates belonged to a common Euro-American genotype, the same as detected in the human case in Guadeloupe. These investigations need to be continued in order to counsel the health authorities about prevention measures, because these recreational thermal baths are used daily by local people and tourists.

Primary amebic meningoencephalitis: a case report and literature review.

Primary amebic meningoencephalitis (PAM) is a rare but nearly always fatal disease caused by infection with Naegleria fowleri, a thermophilic, free-living ameba found in freshwater environments. Cases of N. fowleri infection have been reported from many of the southern-tier states in the United States, with Florida and Texas disproportionately represented among them. Primary amebic meningoencephalitis presents clinically in a fashion that may be indistinguishable from bacterial and viral meningitis. Unfortunately, because the disease is so rare, PAM is often excluded from the differential diagnosis of children with meningitis resulting in delayed diagnostic and therapeutic efforts.Pediatric acute care practitioners in emergency departments, general pediatric wards, and critical care units, especially those practicing in the southern United States, should be familiar with the risk factors for acquisition of PAM, its clinical presentation, and the fact that common empiric treatment of bacterial meningitis will not treat N. fowleri. Herein, we present the case of an adolescent who died of PAM and review the (a) epidemiology, (b) pathophysiology, (c) available diagnostic modalities, (d) treatment options, and (e) outcomes of patients treated for N. fowleri infection of the central nervous system.

Acanthamoeba meningoencephalitis in an immunocompetent patient: an autopsy case report.

Chronic granulomatous CNS infections may be caused by tuberculosis, fungi and rarely by free-living amoeba, especially in immunocompromised individuals. We report a rare, fatal case of granulomatous amoebic encephalitis in an immunocompetent patient mimicking CNS tuberculosis, and review the imageological features and diagnostic tests.

Therapeutic effect of rokitamycin in vitro and on experimental meningoencephalitis due to Naegleria fowleri.

Inhalation of freshwater containing the free-living amoeba Naegleria fowleri leads to a potentially fatal infection known as primary amoebic meningoencephalitis (PAME). Amphotericin B is the only agent with clinical efficacy in the treatment of PAME in humans, however this drug is often associated with adverse effects on the kidney and other organs. In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities of antibacterial agents including clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin and zeocin were examined. Results showed that the growth of amoeba was effectively inhibited by treatment with hygromycin B, rokitamycin and roxithromycin. Notably, when N. fowleri trophozoites were treated with rokitamycin, the minimal inhibitory concentration was 6.25 microg/mL on Day 2. In the treatment of experimental meningoencephalitis due to N. fowleri, survival rates of mice treated with roxithromycin and rokitamycin were 25% and 80%, respectively, over 1 month. The mean time to death for roxithromycin and rokitamycin treatment was 16.2 days and 16.8 days, respectively, compared with 11.2 days for control mice. Finally, rokitamycin showed both in vitro and in vivo therapeutic efficacy against N. fowleri and may be a candidate drug for the treatment of PAME.

Histopathologic spectrum and immunohistochemical diagnosis of amebic meningoencephalitis.

Traditionally, Naegleria fowleri infections are labeled primary amebic meningoencephalitis because of prominent meningeal neutrophilic inflammation. Acanthamoeba spp. and Balamuthia mandrillaris are labeled granulomatous amebic encephalitis because of parenchymal granulomatous inflammation. We compared histopathologic and immunohistochemical features of 18 cases with central nervous system free-living ameba infections. Immunohistochemical assays using polyclonal antibodies that reacted specifically against each genus identified 11 patients with Balamuthia infection, four with N. fowleri, and three with Acanthamoeba. Immunohistochemical assays highlighted the presence of trophozoites that were difficult to identify with hematoxylin and eosin stains in areas of necrosis or where macrophages were abundant. Immunohistochemical assays also demonstrated the presence of granular antigens inside macrophages and blood vessel walls. Amebic cysts were observed in three patients with Acanthamoeba infection and in three with Balamuthia. Patients with Acanthamoeba infection showed granulomatous inflammation. Patients with Naegleria infection had neutrophilic inflammation. Balamuthia infections showed a spectrum of inflammation that ranged from primarily neutrophils to granulomas. Meningitis was present in 88% of cases. Immunohistochemical assays were useful to demonstrate the presence of granular antigens and confirmed the genus of the ameba. The spectrum of inflammation in cases of Balamuthia meningoencephalitis is broader than previously described. The term amebic meningoencephalitis describes better the histopathologic findings than the currently used classification of primary amebic meningoencephalitis and granulomatous amebic encephalitis.

September 2004: a 6-year-old girl with headache and stiff neck.

Free-living amebas in the genera Naegleria, Acanthamoeba and Balamuthia are known to cause CNS infections. Here we report a case of fatal granulomatous amebic meningoencephalitis (GAE) caused by Balamuthia mandrillaris in a 6-year-old previously healthy girl who presented with headache and stiff neck. She was treated medically for brain abscess after a CT scan identified a ring-enhancing lesion in the right temporo-parietal area. A brain biopsy showed necrosis and granulomatous inflammation. Subsequently, multiple new lesions appeared in the brain bilaterally. A second brain biopsy revealed viable amebic trophozoites that were most abundant in perivascular spaces, accompanied by neutrophils, macrophages and eosinophils. Immunofluorescence study confirmed the amoeba as Balamuthia mandrillaris. This case demonstrates that making diagnosis of GAE pre-mortem requires a high index of suspicion. Amebic infection should be included in the differential diagnosis of any granulomatous lesion in CNS; and careful search for amebic parasites should be carried out especially when necrosis predominates in the pathological material.