Current Research on Free-living Amebae Causing Granulomatous Amebic Encephalitis
Investigators studying free-living amebae were invited to participate in the VIIth International Workshops on Opportunistic Protists in order to increase awareness of, and interactions with, other investigators studying opportunistic protists. Seven papers or posters on free-living amebae that are opportunistic pathogens were presented at the meeting. Presentations on free-living amebae are summarized below.
To date, two genera of free-living amebae have been associated with fatal opportunistic infections of the central nervous system in humans. Acanthamoeba spp. and Batamuthia mandrillaris are the causative agents of Granulomatous Amebic Encephalitis (GAE), a progressive fatal disease of the brain in immunocompromised individuals. Although GAE is rare, there has been an increase in the reported number of persons afflicted worldwide. This may be due to an increase in the number of patients with Acquired Immune Deficiency Syndrome, of individuals undergoing cancer chemotherapy, and/or of patients with underlying or debilitating diseases such as Systemic Lupus Erythematosus, diabetes, or alcoholism. Alternatively, the perceived increase in the incidence may be due to greater awareness of these organisms as opportunistic pathogens. One difficulty noted in free-living amebic infections is the lack of specific methods to detect and identify the causative organisms, and the lack of differential diagnostic signs clinically. Most infections are recognized post-mortem.
Allen (Indiana University School of Medicine) reported that most clinical laboratories do not offer methods to diagnose free-living amebae infections, and pathologists often do not recognize amebae in tissue sections. His studies demonstrated a need for the development of a rapid, sensitive, and reliable method to diagnose free-living ameba infections, which are almost always fatal. The pathologic aspects of infections by Acanthamoeba and Naegleria in experimental animals were compared with pathologic findings in human infections. Infection with Acanlhamoeba via the nasal passages results in amebae invading the nasal epithelium and entering the brain through openings in the cribriform plate leading amebae into the subarachnoid plate. An inflammatory response occurs and abscesses or granutamata form in the gray matter around the amebae. Many of the pathologic changes in brain reported for experimental animals also occur in human infections. (PL28- S. Allen. Acanthamoeba and Naegleria: Pathologic aspects of infections in humans and other hosts).
Identification of Acanthamoeba at the genus level is relatively easy because of the distinctive morphological characteristics of the cyst and the appearance of spiny acanthapodia on trophozoites. Since identification of Acanthamoeba at the species level is difficult and a number of different species may be the causative agents of GAE, the Byers group (Ohio State University) described the use of an Acanthamoeba DNA Database for identification of clinical and environmental samples of Acanthamoeba. A DNA database of Acanthamoeba nuclear and mitochondrial sequences has been developed at Ohio State. The database currently contains more than 130 nuclear ribosomal genes, 65 mitochondrial genes and 6 mitochondrial spacer regions. The use of these sequences for detection and identification of organisms is being evaluated. The reliability of the 18S rDNA sequences of acanthamoebae for identifying clinical and environmental isolates was examined using Acanthamoeba isolated from corneal scrapes, contact lens cases, and home water supplies of patients with Acanthamoeba kerititis (AK). Booton and colleagues (Ohio State University) presented evidence that the T4 genotype is preferentially associated with AK. PO11 - G. Booton, T. Byers, D. Kelly, P Fuerst. The use of the Acanthamoeba DNA
Database for the identification of clinical and environmental samples of Acanthamoeba. PO13 - D. Kelly, G. Booton, Y. W. Chu, D. Seal, E. Houang, P. Fuerst, T. Byers. 18S rDNA sequence typing of amoebae from corneal scrapes, contact lens cases and home water supplies of Acanthamoeba kerititis patients in Hong Kong.)
A presentation by Marciano-Cabral and colleagues (Medical College of Virginia Commonwealth University) demonstrated exacerbation of brain infections with Acanthamoeba in experimental animals treated with delta-9-Tetrahydrocannabinol (THC), the major psychoactive and major immunosuppressive component of marijuana. Higher mortalities were observed in mice infected with Acanthamoeba castellanii and treated with THC than in infected drug-free mice. Additionally, THC treatment decreased the time to death. THC is thought to act by inhibiting microglial cell migration to areas of infection and by inhibiting microglial cells (brain macrophages) from acting as effectors against the amebae. Because marijuana is immunosuppressive and there is a movement to permit use of marijuana for medicinal purposes in AIDS patients and cancer patients, the need to examine the effects of THC on infections with opportunistic pathogens is pressing. (PL6 - F. Marciano-Cabral, S.G. Bradley, G. A. Cabral. Delta-9-tetrahydrocannabinol (THC) the major psychoactive component of marijuana exacerbates brain infection by Acanthamoeba).
Newsome and colleagues (Middle Tennessee State University) demonstrated that free-living amebae can serve as host cells for Legionella pneumophila. Legionella use Acanthamoeba for intracellular replication. A 17,000 fold increase in L. pneumophila colony forming units was observed after coculture with Acanthamoeba polyphaga. The amebae are subsequently lysed. This study also describes the isolation and characterization of previously undescribed bacteria observed in amebae from environmental sources. Newsome suggested that these bacteria in association with free-living amebae may be responsible for some as yet unidentified causes of respiratory diseases. (PL102 - A. L. Newsome, M. B. Farone, and S. B. Berk. Free-living amoebae as opportunistic hosts for intracellular bacterial parasites).
A more recently recognized opportunistic free-living ameba, Balamuthia mandrillaris, has been found to cause GAE. Martínez (University of Pittsburgh) discussed case studies and pathologic consequences of this fatal central nervous system disease. Balamuthia causes a chronic fatal hemorrhagic necrosis of the brain. GAE cases caused by Balamuthia have only been diagnosed post-mortem. No environmental niche has been identified and the means of exposure or route of infection of humans remains unknown. (PL103 - A. J. Martínez, F. L. Schuster, G. S. Visvesvara. Balamuthia mandrillaris: Its pathogenic potential).
A serologic survey of humans with encephalitis, conducted as part of the California Encephalitis Project, was reported by F. Schuster and colleagues. From 130 encephalitis patients, three samples with high titers of antibody were detected, as measured by immunoflourescence assays. Titers of sera between 2 to 32 were considered negative, while positive titers ranged from 32 – 2048. This study indicated that Balamuthia infections can be diagnosed by serological assays but more rapid tests are needed to identify these organisms in human infection. (PL 22 - F. L. Schuster, C. Glaser, S. Gilliam, G. S. Visvesvara. Survey of sera from encephalitis patients for Balamuthia mandrillaris antibody).
In conclusion, the majority of presentations focused on the pathology of GAE infections caused by Acanthamoeba or Balamuthia. The presentations at the present workshop advanced our knowledge of the pathology and methods of identification of the organisms. Much remains to be learned concerning the cellular and molecular biology of the organisms that cause Granulomatous Amebic Encephalitis.
