Background: Free-living amoebas (FLAs) can cause severe and fatal central nervous system infections that are difficult to diagnose. Methods: We present the case of a 74-year-old immunocompetent woman admitted for focal neurological symptoms with enhancing lesions in the right cerebellar hemisphere. A first cerebral biopsy showed granulomatous inflammation, but no microorganisms were identified. After transient clinical improvement, she eventually deteriorated 4 months after initial presentation, with an MRI confirming multiple new masses affecting all cerebral lobes. Results: A second brain biopsy revealed granulomatous and acute inflammation with organisms containing a large central nucleus with prominent karyosome, consistent with FLAs. Immunohistochemical and polymerase chain reaction assays performed at CDC were positive for Acanthamoeba spp, confirming the diagnosis of granulomatous amoebic encephalitis (GAE) caused by Acanthamoeba spp. The patient was treated with combination therapy recommended by CDC, but died a few days later. Upon histopathological rereview, amoebic cysts and trophozoites were identified by histochemical and immunohistochemical methods in the first cerebral biopsy. Conclusion: FLA infections can be challenging to diagnose because of the low incidence, non-specific clinical and radiological presentation, lack of accessible diagnostic tools, and clinicians' unfamiliarity. This case highlights the importance of recognizing FLA as a potential cause of granulomatous encephalitis, even in the absence of risk factors, as early treatment might be associated with favourable outcomes in case reports. When suspected, CDC laboratories offer tests to confirm the diagnosis promptly. | Historique : Les amibes libres peuvent causer des infections du système nerveux central graves et fatales qui sont difficiles à diagnostiquer. Méthodologie : Les auteurs présentent le cas d'une femme immunocompétente de 74 ans hospitalisée à cause de symptômes neurologiques focaux avec lésions rehaussantes dans l'hémisphère cérébelleux droit. Une première biopsie cérébrale a révélé une inflammation granulomateuse, mais aucun microorganisme n'a été décelé. Après une amélioration clinique transitoire, son état s'est détérioré quatre mois après la première consultation, et l'IRM a confirmé de multiples nouvelles masses touchant tous les lobes cérébraux. Résultats : Une deuxième biopsie cérébrale a révélé une inflammation granulomateuse aiguë par des organismes dont les gros noyaux centraux et les caryosomes volumineux étaient évocateurs d'amibes libres. L'immunohistochimie et l'amplification en chaîne par polymérase effectuées aux CDC se sont avérés positives pour Acanthamoeba spp, ce qui a confirmé un diagnostic d'encéphalite amibienne granulomateuse causée par Acanthamoeba spp. La patiente a reçu une polythérapie recommandée par les CDC, mais est malheureusement décédée quelques jours plus tard. À la reprise de l'analyse histopathologique, des kystes amibiens et des trophozoïtes ont été décelés dans la première biopsie cérébrale par des méthodes histochimiques et immunohistochimiques. Conclusion : Les infections par des amibes libres peuvent être difficiles à diagnostiquer en raison de leur faible incidence, de leur présentation clinique et radiologique non spécifique, de l'absence d'outils diagnostiques accessibles et de la méconnaissance des cliniciens. Ce cas renforce l'importance d'inclure les amibes libres dans les causes potentielles d'encéphalite granulomateuse, même en l'absence de facteurs de risque, car un traitement rapide a été associé à des résultats favorables dans certains rapports de cas. Lorsqu'on en soupçonne la présence, les laboratoires des CDC offrent des tests pour confirmer rapidement le diagnostic.

We present the chromosome sequences of a Naegleria fowleri isolate from a human primary amebic meningoencephalitis (PAM) case. The genome sequences were assembled from Illumina HiSeq and PacBio sequencing data and verified with the optical mapping data. This led to the identification of 37 contigs representing 37 chromosomes in N. fowleri.

There are over forty species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multi-copy SSU rDNA sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as few as 0.1 trophozoite-equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in the mixed infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated comparable sensitivity and specificity with that of the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly-developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically-identical Entamoeba species especially in low resource settings.

Primary amebic meningoencephalitis (PAM), caused by the free-living ameba Naegleria fowleri, has a fatality rate of over 97%. Treatment of PAM relies on amphotericin B in combination with other drugs, but few patients have survived with the existing drug treatment regimens. Therefore, development of effective drugs is a critical unmet need to avert deaths from PAM. Since ergosterol is one of the major sterols in the membrane of N. fowleri, disruption of isoprenoid and sterol biosynthesis by small-molecule inhibitors may be an effective intervention strategy against N. fowleri. The genome of N. fowleri contains a gene encoding HMG-CoA reductase (HMGR); the catalytic domains of human and N. fowleri HMGR share <60% sequence identity with only two amino acid substitutions in the active site of the enzyme. Considering the similarity of human and N. fowleri HMGR, we tested well-tolerated and widely used HMGR inhibitors, known as cholesterol-lowering statins, against N. fowleri. We identified blood-brain-barrier-permeable pitavastatin as a potent amebicidal agent against the U.S., Australian, and European strains of N. fowleri. Pitavastatin was equipotent to amphotericin B against the European strain of N. fowleri; it killed about 80% of trophozoites within 16 h of drug exposure. Pretreatment of trophozoites with mevalonate, the product of HMGR, rescued N. fowleri from inhibitory effects of statins, demonstrating that HMGR of N. fowleri is the target of statins. Because of the good safety profile and availability for both adult and pediatric uses, consideration should be given to repurposing the fast-acting pitavastatin for the treatment of PAM.

BACKGROUND: Amoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential to limiting ocular morbidity. We undertook a quality improvement initiative around the molecular detection of Acanthamoebae in our laboratory due to an unusual case of discordance. METHODS: Nine ATCC strains of Acanthamoeba and 40 delinked, biobanked surplus corneal scrapings were retrieved and analyzed for the presence of Acanthamoebae by 4 separate real time PCR assays. The assay used by the Free Living and Intestinal Amebas laboratory of the CDC was considered the reference standard, and performance characteristics of each individual assay, and pairs of assays were calculated. Outcome measures were sensitivity, specificity, and positive predictive value (PPV) and negative (NPV) predictive value. RESULTS: Of 49 included specimens, 14 (28.6%) were positive by the gold standard assay, and 35 (71.4%) were negative. Sensitivity of the individual test assays ranged from 64.3% to 92.9% compared to the gold standard, while specificity ranged from 88.6% to 91.4%. PPV and NPV ranged from 69.2% to 78.6%, and 86.1% to 96.9%, respectively. Combinations of assay pairs led to improved performance with sensitivities ranging from 92.9% to 100%, and specificity from 97.1% to 100%. ATCC and clinical strains of Acanthamoeba that failed detection by certain individual assays included A. castellani, A. culbertsoni, and A. lenticulata. In 3 clinical specimens, false negativity of the gold standard assay could not be excluded. CONCLUSIONS: Molecular diagnostic approaches, especially combinations of both highly sensitive and specific assays, offer a reasonably performing, operator independent, and rapid strategy for the detection of Acanthamoebae from clinical specimens, and are likely more practical than either culture or direct microscopic detection.

Among the Entamoeba species that infect humans, Entamoeba histolytica causes diseases, Entamoeba dispar is a harmless commensal, Entamoeba moshkovskii seems to be a pathogen, and the pathogenicity of Entamoeba bangladeshi remains to be investigated. Species-specific detection needed for treatment decisions and for understanding the epidemiology and pathogenicity of these amebae. Antigen-based detection methods are needed for E dispar, E moshkovskii, and E bangladeshi; and molecular diagnostic test capable of detecting E histolytica, E dispar, E moshkovskii, and E bangladeshi simultaneously in clinical samples. Next-generation sequencing of DNA from stool is needed to identify novel species of Entamoeba.