Treatment Of Primary Amoebic Meningoencephalitis Research Articles

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 μM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 μM) while the reported CC50 was >300 μM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.

Development and immunological evaluation of an mRNA-based vaccine targeting Naegleria fowleri for the treatment of primary amoebic meningoencephalitis

More than 95% of patients fall victim to primary amoebic meningoencephalitis (PAM), a fatal disease attacking the central nervous system. Naegleria fowleri, a brain-eating microorganism, is PAM's most well-known pathogenic ameboflagellate. Despite the use of antibiotics, the fatality rate continues to rise as no clinical trials have been conducted against this disease. To address this, we mined the UniProt database for pathogenic proteins and selected assumed epitopes to create an mRNA-based vaccine. We identified thirty B-cell and T-cell epitopes for the vaccine candidate. These epitopes, secretion boosters, subcellular trafficking structures, and linkers were used to construct the vaccine candidate. Through predictive modeling and confirmation via the Ramachandran plot (with a quality factor of 92.22), we assessed secondary and 3D structures. The adjuvant RpfE was incorporated to enhance the vaccine construct's immunogenicity (GRAVY index: 0.394, instability index: 38.99, antigenicity: 0.8). The theoretical model of immunological simulations indicated favorable responses from both innate and adaptive immune cells, with memory cells expected to remain active for up to 350 days post-vaccination, while the antigen was eliminated from the body within 24 h. Notably, strong interactions were observed between the vaccine construct and TLR-4 (− 11.9 kcal/mol) and TLR-3 (− 18.2 kcal/mol).

Cyanomethyl Vinyl Ethers Against Naegleria fowleri.

Naegleria fowleri is a pathogenic amoeba that causes a fulminant and rapidly progressive disease affecting the central nervous system called primary amoebic meningoencephalitis (PAM). Moreover, the disease is fatal in more than 97% of the reported cases, mostly affecting children and young people after practicing aquatic activities in nontreated fresh and warm water bodies contaminated with these amoebae. Currently, the treatment of primary amoebic meningoencephalitis is based on a combination of different antibiotics and antifungals, which are not entirely effective and lead to numerous side effects. In the recent years, research against PAM is focused on the search of novel, less toxic, and fully effective antiamoebic agents. Previous studies have reported the activity of cyano-substituted molecules in different protozoa. Therefore, the activity of 46 novel synthetic cyanomethyl vinyl ethers (QOET-51 to QOET-96) against two type strains of N. fowleri (ATCC 30808 and ATCC 30215) was determined. The data showed that QOET-51, QOET-59, QOET-64, QOET-67, QOET-72, QOET-77, and QOET-79 were the most active molecules. In fact, the selectivity index (CC50/IC50) was sixfold higher when compared to the activities of the drugs of reference. In addition, the mechanism of action of these compounds was studied, with the aim to demonstrate the induction of a programmed cell death process in N. fowleri.

Repurposing in vitro approaches for screening anti-parasitic drugs against the brain-eating amoeba Naegleria fowleri

Naegleria fowleri is both a pathogenic and a free-living microbial eukaryote, responsible for the development of primary amoebic meningoencephalitis (PAM) in humans. PAM is a rapid, severe and fatal underestimated infectious disease, which has been reported in countries with warmer climates. The major drawbacks with PAM are the lack of effective therapies and delay in diagnosis. The current frontline treatment presents a low rate of recovery (5%) and severe adverse effects. For example, many drug candidates lack efficacy, since they do not effectively cross the blood-brain-barrier. Consequently, more effective drugs are urgently needed. Herein, we report a new in vitro method suitable for medium- and high-throughput drug discovery assays, using the closely related Naegleria gruberi as a model. We have subsequently used this method to screen a library of 1175 Food and Drug Administration-approved drugs. As a result, we present three drugs (camptothecin, pyrimethamine, and terbinafine) that can be repurposed, and are anticipated to readily cross the blood-brain-barrier with activity against Naegleria species in therapeutically achievable concentrations. Successively, we integrated several in vitro assays that resulted in identifying fast-acting and high amoebicidal drugs. In conclusion, we present a new approach for the identification of anti-Naegleria drugs along with three potential drug candidates for further development for the treatment of PAM.

Drugs used for the treatment of cerebral and disseminated infections caused by free-living amoebae.

Free‐living amoebae (FLAs) are protozoa developing autonomously in diverse natural or artificial environments. The FLAs Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri represent a risk for human health as they can become pathogenic and cause severe cerebral infections, named granulomatous amoebic encephalitis (GAE), Balamuthia amoebic encephalitis (BAE), and primary amoebic meningoencephalitis (PAM), respectively. Additionally, Acanthamoeba sp. can also rarely disseminate to diverse organs, such as the skin, sinuses, or bones, and cause extracerebral disseminated acanthamebiasis (EDA). No consensus treatment has been established for cerebral FLA infections or EDA. The therapy of cerebral and disseminated FLA infections often empirically associates a large diversity of drugs, all exhibiting a high toxicity. Nevertheless, these pathologies lead to a high mortality, above 90% of the cases, even in the presence of a treatment. In the present work, a total of 474 clinical cases of FLA infections gathered from the literature allowed to determine the frequency of usage, as well as the efficacy of the main drugs and drug combinations used in the treatment of these pathologies. The efficacy of drug usage was determined based on the survival rate after drug administration. The most efficient drugs, drug combinations, and their mechanism of action were discussed in regard to the present recommendations for the treatment of GAE, EDA, BAE, and PAM. At the end, this review aims to provide a useful tool for physicians in their choice to optimize the treatment of FLA infections.

Oleic Acid Coated Silver Nanoparticles Showed Better in Vitro Amoebicidal Effects against Naegleria fowleri than Amphotericin B.

Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis (PAM) which almost always results in death. N. fowleri is also known as "brain-eating amoeba" due to its literal infestation of the brain leading to an inflammatory response in the brain tissues. Currently, there is no single drug that is available to treat PAM, and most treatments are combinations of antifungal, anticancer, and anti-inflammatory drugs. Recently nanotechnology has gained attention in chemotherapeutic research converging on drug delivery, while oleic acid (OA) has shown positive effects on the human immune system and inflammatory processes. In continuation of our recent research in which we reported the effects of oleic acid conjugated with silver nanoparticles (OA-AgNPs) against free-living amoeba Acanthamoeba castellanii, in this report, we show their antiamoebic effects against N. fowleri. OA alone and its nanoconjugates were tested against the amoeba by using amoebicidal and host cell cytopathogenicity assays. Trypan blue exclusion assay was used to determine cell viability. The results revealed that OA-AgNPs exhibited significantly enhanced antiamoebic effects (P < 0.05) against N. fowleri as compared to OA alone. Evidently, lactate dehydrogenase release shows reduced N. fowleri-mediated host cell cytotoxicity. Based on our study, we anticipate that further studies on OA-AgNPs could potentially provide an alternative treatment of PAM.

Phenotypic Screens Reveal Posaconazole as a Rapidly Acting Amebicidal Combination Partner for Treatment of Primary Amoebic Meningoencephalitis.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration-approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 μM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri-infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.

Effect of DNA Vaccination with Retroviral Vector Expressing nfa1 Gene in Naegleria fowleri Infection

Naegleria fowleri is pathogenic free-living amoeba leading to primary amoebic meningoencephalitis(PAM) in human and animals. The nfa1 gene cloned from N. fowleri is located on food-cup structure called pseudopodia and function an adherence of host target cells. To evaluate the effect of nfa1 vaccination against N. fowleri infection, we constructed retroviral vector(pQCXIN) expressing nfa1 gene. To determine the effect of vaccination and protective immunity in in vivo models, we measured the immunoglobulin levels, cytokine induction, and survival rate in mice infected with N. fowleri. Both levels of IgG and IgG subclass in DNA vaccinated mice were significantly elevated. The cytokine analysis show that DNA vaccinated mice induces production of IL-4 and IFN-γcytokines suggesting a Th1/Th2 mixed type immune response. The levels of nfa1 specific IgG antibody were maintained highly until 12 weeks post-vaccination in vaccinted mice. The nfa1 vaccinated mice using retroviral vector increased significantly survival rate(60%) after N. fowleri infection. Consequently, the nfa1 vaccination effectively induces protective immunity by upregulation of immune response in mice infected with N. fowleri. These results suggest that DNA vaccination using retroviral vector may be proper trial for treatment and prevention of PAM.

CYP51 is an essential drug target for the treatment of primary amoebic meningoencephalitis (PAM).

Primary Amoebic Meningoencephalitis (PAM) is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered “rare” (but likely underreported) the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug ‘repurposing’—a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51) target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i) systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii) identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii) analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially ‘druggable’ target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.

Naegleria fowleri: Diagnosis, Pathophysiology of Brain Inflammation, and Antimicrobial Treatments.

Primary amoebic meningoencephalitis (PAM) is a very rare disease with a high mortality rate. PAM is caused by Naegleria fowleri, an amoeba which resides in freshwater lakes and ponds and can survive in inadequately chlorinated pools ( Lopez, C.; Budge, P.; Chen, J., et al. Primary amebic meningoencephalitis: a case report and literature review . Pediatr. Emerg. Care 2012 , 28 , 272 - 276 ). In the past 50 years, there have been over 130 cases of Naegleria induced PAM in the United States with only three known survivors; one survivor was diagnosed and treated at Arkansas Children's Hospital. Successful treatment of PAM started with a rapid diagnosis, extensive antimicrobial therapy including an investigational medication miltefosine, supportive care, an intraventricular shunt, and hypothermia. These treatments address different aspects of the disease process. Increased understanding of the diagnosis and treatment of PAM is important especially for patients who present with meningitis-like findings during the summer months.

Successful treatment of a case of primary amoebic meningoencephalitis: How important is history taking.

Sir, Naegleria fowleri is a rare cause of meningoencephalitis leading to a fatal fulminant primary amoebic meningoencephalitis (PAM) acquired after diving, swimming in warm fresh water. Very few patients are reported to survive after acquiring this infection.[1,2] Timely diagnosis requires a high index of suspicion in any individual presenting with a sub-acute onset meningitis after exposure to standing water in lakes and pools. A 6-year-old male child presented with a history of high grade fever, headache, vomiting for 10 days and altered sensorium for 1-day. There was no history of seizures, any other complaints or contact with tuberculosis. There was a history of playing and swimming in water stored in a cement tank, collected from a stream of water (Koohl). On examination, the child was in delirium, febrile with stable vitals. Central nervous system (CNS) examination revealed signs of meningeal irritation with the early papilledema. Rest of the systemic examination was normal. On admission possibilities of acute pyogenic meningitis, rickettsial meningoencephalitis, as this region is endemic for scrub typhus and tubercular meningitis, was kept. Intravenous (IV) ceftriaxone, azithromycin, mannitol, maintenance fluid and supportive care were started. Cerebrospinal fluid (CSF) analysis after lumbar puncture that was done next day, showed 415 cells/mm3 with 30% neutrophils and 70% lymphocytes. Gram-staining did not reveal any bacteria, but the wet mount smear showed plenty of motile, amoeboid flagellate organisms, 15-20 μm in size, Naegleria species. The water that was later on collected from water tank and koohl also showed similar Naegleria. CSF culture was negative for bacteria and computed tomography head done on 2nd day showed mild cerebral edema. The child was started on IV amphotericin B (1 mg/kg/d IV for 21 days), fluconazole (12 mg/kg/d PO on day 1 followed by 6 mg/kg/day for total of 21 days), oral rifampicin (15 mg/kg/d PO for 21 days) and supportive care for 3 weeks. The child showed gradual improvement with normal sensorium in 3 days, afebrile and disappearance of neck rigidity after 6 and 8 days of treatment respectively. He was discharged and followed-up for 3 months with no residual CNS abnormality. Primary amoebic meningoencephalitis is a fulminant and rapidly fatal disease that principally affects children and young adults caused by N. fowleri, an amoeba-flagellate found in most soil and freshwater habitats. Most of the cases reported in children as well as in adults has been fatal.[1,3] One of the key and most important factors for a successful outcome is the promptness in diagnosis and treatment of the patient. Delay in diagnosis and the fulminant nature of the disease results in few survivors.[4] In our case, the child presented with features of meningitis and altered sensorium with sub-acute course with history of swimming in stored fresh water. In a case of meningitis, we routinely do not perform wet mount microscopy but in our case history of swimming in stored fresh water with a sub-acute course of meningoencephalitis led to the investigation and early diagnosis, which is the most important factor for successful treatment of PAM. Early treatment was started, and the child recovered completely. Vargas-Zepeda et al.[2] also successfully treated a 10-year-old boy of PAM with IV amphotericin B, fluconazole, rifampicin, and dexamethasone. So, history taking that is perhaps a dwindling art can create a difference perhaps between death and survival and wet mount microscopy should be routinely done in a case of meningitis or meningoencephalitis with sub-acute course.

Diagnosis, Clinical Course, and Treatment of Primary Amoebic Meningoencephalitis in the United States, 1937-2013

Primary amoebic meningoencephalitis (PAM) is a rapidly progressing waterborne illness that predominately affects children and is nearly always fatal. PAM is caused by Naegleria fowleri, a free-living amoeba found in bodies of warm freshwater worldwide. We reviewed exposure location, clinical signs and symptoms, diagnostic modalities, and treatment from confirmed cases of PAM diagnosed in the United States during 1937-2013. Patients were categorized into the early (ie, flu-like symptoms) or late (ie, central nervous system signs) group on the basis of presenting clinical characteristics. Here, we describe characteristics of the survivors and decedents. The median age of the patients was 12 years (83% aged ≤18 years); males (76%) were predominately affected (N = 142). Most infections occurred in southern-tier states; however, 4 recent infections were acquired in northern states: Minnesota (2), Kansas (1), and Indiana (1). Most (72%) of the patients presented with central nervous system involvement. Cerebrospinal fluid analysis resembled bacterial meningitis with high opening pressures, elevated white blood cell counts with predominantly neutrophils (median, 2400 cells/μL [range, 5-26 000 cells/μL]), low glucose levels (median, 23 mg/dL [range, 1-92 mg/dL]), and elevated protein levels (median, 365 mg/dL [range, 24-1210 mg/dL]). Amoebas found in the cerebrospinal fluid were diagnostic, but PAM was diagnosed for only 27% of the patients before death. Imaging results were abnormal in approximately three-fourths of the patients but were not diagnostic for amoebic infection. Three patients in the United States survived. To our knowledge, this is the first comprehensive clinical case series of PAM presented in the United States. PAM is a fatal illness with limited treatment success and is expanding into more northern regions. Clinicians who suspect that they have a patient with PAM should contact the US Centers for Disease Control and Prevention at 770-488-7100 (available 24 hours/day, 7 days/week) to discuss diagnostic testing and treatment options (see cdc.gov/naegleria).

Effects of trifluralin on growth and differentiation of the amoebo-flagellate Naegleria

The microtubule-disrupting herbicide trifluralin (TFL) inhibits growth and flagellate transformation of the amoebo-flagellates Naegleria fowleri and N. gruberi. Growth is less sensitive than is flagellar transformation to TFL: 50 μM reversibly arrests growth while 10 μM irreversibly blocks transformation. Naegleria shows a high sensitivity to TFL like trypanosomatids and unlike the low sensitivity to mammals. TFL is potentially useful for the study of flagellate transformation especially since colchicine does not effect Naegleria. It is conceivable that TFL may be useful in the treatment of primary amoebic meningoencephalitis caused by N. fowleri.

Effects of trifluralin on growth and differentiation of the amoebo-flagellateNaegleria

The microtubule-disrupting herbicide trifluralin (TFL) inhibits growth and flagellate transformation of the amoebo-flagellates Naegleria fowleri and N. gruberi. Growth is less sensitive than is flagellar transformation to TFL: 50 μM reversibly arrests growth while 10 μM irreversibly blocks transformation. Naegleria shows a high sensitivity to TFL like trypanosomatids and unlike the low sensitivity to mammals. TFL is potentially useful for the study of flagellate transformation especially since colchicine does not effect Naegleria. It is conceivable that TFL may be useful in the treatment of primary amoebic meningoencephalitis caused by N. fowleri.

Successful treatment of amoebic meningoencephalitis in a Chinese living in Hong Kong

Primary amoebic meningoencephalitis due to Naegleria fowleri was found in a 38-year-old Chinese man living in Hong Kong who presumably acquired the infection from swimming in a hot spring in neighbouring China. Amoebic cysts were identified in tissue taken from a brain abscess. The patient responded to surgical drainage and a 6-week course of amphotericin B, rifampicin and chloramphenicol. This is one of 6 cases of successful treatment of primary amoebic meningoencephalitis documented in the medical literature.

Treatment of Experimental Naegleria Meningoencephalitis with a Combination of Amphotericin B and Rifamycin

The treatment of primary amoebic meningoencephalitis was examined using a mouse model. Rifamycin by itself was ineffective. However, a synergistic effect was observed when used in combination with amphotericin B. This finding may prove useful in the treatment of patients with primary amoebic meningoencephalitis, for which satisfactory treatment is currently unavailable.

Synergism between tetracycline and amphotericin B in experimental amoebic meningoencephalitis.

The efficacy of tetracycline and amphotericin B in the treatment of primary amoebic meningoencephalitis was studied by means of a mouse model. The results showed marked synergism between these two drugs. Mice who recieved 50 microgram amphotericin B daily succumbed to the infection with only 28.6% survival compared to 0% in untreated controls. In contrast, survival percentage was increased to 66.7% and 84.6% in mice receiving the combined dosage schedule of 50 microgram amphotericin B with 2 tetracycline, and 50 microgram amphotericin B with 3 mg tetracycline, respectively. The finding of synergism between tetracycline and amphotericin may provide a better means for the treatment of this disease.