Govinda S. Visvesvara

The epidemiology of Acanthamoeba keratitis in the United States

We surveyed members of the Ocular Microbiology and Immunology Group and reviewed laboratory requests at the Centers for Disease Control to determine better the epidemiology of Acanthamoeba keratitis in the United States. A total of 208 cases of Acanthamoeba keratitis were identified. The number of cases increased gradually between 1981 and 1984, with a dramatic increase beginning in 1985. Males and females were equally affected. Of the 208 patients, 85 (41%) resided in California, Texas, Florida, or Pennsylvania. Of 189 patients, 160 (85%) wore contact lenses, predominantly daily-wear or extended-wear soft lenses. Of the 138 patients who wore contact lenses and for whom information was available, 88 (64%) used saline prepared by dissolving salt tablets in distilled water. Patients aged 50 years and older were more likely to have had a history of trauma than younger patients, and males were more likely to have a history of trauma than females.

Pathologic Features and Immunofluorescent Antibody Demonstration of Ocular Microsporidiosis (Encephalitozoon hellem) in Seven Patients with Acquired Immunodeficiency Syndrome

We studied the clinicopathologic features of seven patients with acquired immunodeficiency syndrome (AIDS) and ocular microsporidiosis. All patients had decreased levels of CD4-positive cells (mean, 26/ml3) and ocular symptoms; five had bilateral punctate epithelial keratopathy, one had intermittent red eyes with conjunctivitis, and one had red eyes only. Light and electron microscopy of corneal and conjunctival biopsy and cytologic specimens and intact globes disclosed microsporidia belonging to the genus Encephalitozoon. Because E. cuniculi and E. hellem, the two species of the Encephalitozoon genus, are morphologically identical, an immunofluorescent antibody technique was used for species identification. In all seven patients, the agent was identified as E. hellem. Pathologic examination of globes obtained after autopsy disclosed E. hellem infection to be restricted to the corneal and conjunctival epithelium. We studied methods for the routine diagnosis of ocular microsporidiosis in patients with AIDS, including the role of immunofluorescent antibody staining.

Short‐Term In Vitro Culture and Molecular Analysis of the Microsporidian, Enterocytozoon bieneusi

ABSTRACT. The microsporidium, Enterocytozoon bieneusi, causes a severe, debilitating, chronic diarrhea in patients with the acquired immunodeficiency syndrome. Specific diagnosis of intestinal microsporidiosis, especially due to Enterocytozoon, is difficult and there is no known therapy that can completely eradicate this parasite. Preliminary studies indicate that a short term (about 6 months) in vitro culture of this parasite yielding low numbers of spores, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients. The cultures may subsequently be used for the isolation and molecular analysis of parasite DNA.

Granulomatous amebic encephalitis: a review and report of a spontaneous case from Venezuela

Granulomatous amebic encephalitis (GAE), or meningoencephalitis due to Acanthamoeba spp. and leptomyxid ameba are uncommon CNS infections that generally occur in immunocompromised hosts. We describe a case of GAE caused by Balamuthia mandrillaris previously designated as a leptomyxid ameba, in an apparently healthy 14-year-old Venezuelan boy. This case was characterized by sudden onset of seizures, focal neurologic signs and by a prolonged clinical course (from November 1992 to March 1993). Neuroimaging studies showed cerebral hypodense lesions in cerebral hemispheres, brain stem and cerebellum. Microscopically, we found a chronic granulomatous inflammatory reaction with necrotizing angiitis, large numbers of amebic trophozoites and few cysts in perivascular spaces and within necrotic CNS tissue. The amebas were identified as B. mandrillaris based on their immunofluorescence reactivity with the anti-B. mandrillaris serum. So far, 30 cases of GAE due to B. mandrillaris have been recognized in humans, two in AIDS patients. No visceral involvement by free-living amebas or any other significant abnormality was observed. This patient developed “spontaneous” GAE, but it remains possible that an undiagnosed abnormality in cell-mediated immunity or a deficient humoral immune response may explain the susceptibility of this patient to this opportunistic infection.

Inhibition of the spore polar filament extrusion of the microsporidium, Encephalitozoon hellem, isolated from an AIDS patient.

Spores of the microsporidian parasitic protozoan Encephalitozoon hellem were purified and incubated at 37° C in a solution with an electrolyte composition similar to that of mammalian extracellular fluid, and in solution in which the calcium had been replaced with 0.2 mM EGTA. Polar filament extrusion (germination) was monitored by both scanning electron microscopy and light microscopy. Germination was pH‐dependent, with optima at pH 7.4 and 9.5, and was significantly greater in the presence of medium calcium. Hydrogen peroxide caused a concentration‐dependent increase in germination that was also reduced in a calcium‐free medium. Four agents were found to inhibit spontaneous and H2O2‐stimulated polar filament extrusion: the microfilament disrupter, cytochalasin D; the microtubule disrupter, demecolcine; the calcium channel blocker, nifedipine; and the antifungal agent, itraconazole. These results are consistent with the existence of a calcium‐channel‐mediated step, and requirements for an F‐actin‐ and for a tubulin‐containing element in the germination process of the spore of this parasite. Nifedipine, cytochalasin D and itraconazole all have different sites of action and were therefore able to potentiate one another when used in paired combination to inhibit germination.

Asymptomatic Respiratory Tract Microsporidiosis Due to Encephalitozoon hellem in Three Patients with AIDS

Microsporidia of the genera Enterocytozoon and Encephalitozoon have been identified as frequent causes of intestinal and disseminated infections, respectively, in patients with AIDS. Even though most subjects infected with these protozoa develop overt disease, simple colonization without illness may occur, as we observed in three severely immunosuppressed patients with AIDS. The parasites, recognized in and isolated from bronchoalveolar lavage sediment specimens, were characterized as Encephalitozoon hellem. Colonization of the bronchial tree was temporary, and treatment with albendazole was not needed to clear the infection.

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

BACKGROUNDnPrimary 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.nnnMETHODSnWe 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.nnnRESULTnThe 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.nnnCONCLUSIONSnTo 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).

Calcium and hydrogen ion concentrations in the parasitophorous vacuoles of epithelial cells infected with the microsporidian Encephalitozoon hellem.

ABSTRACT. Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura‐2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.

Isolation and identification of Encephalitozoon hellem from an Italian AIDS patient with disseminated microsporidiosis.

Microsporidia are primitive mitochondria‐lacking spore‐forming eukaryotic protozoa that infect a wide variety of animals and also humans. Of the five genera (Encephalitozoon, Enterocytozoon, Septula, Nosema and Pleistophora) that cause infections in humans, Enterocytozoon bieneusi. Septula intestinulis, and Encephulitozoon hellem are being increasingly identified in patients with acquired immunodeficiency syndrome (AIDS). E. bieneusi causes gastrointestinal disease, S. intestinulis causes gastrointestinal and disseminated disease, and E. hellem causes ocular as well as disseminated disease. We have established in continuous culture a strain of microsporidia isolated from the urine and throat washings of an Italian AIDS patient and identified it as Encephalitozoon hellem, based on its ultrastructural morphology, antigenic pattern, and polymerase chain reaction‐amplified small subunit ribosomal RNA. We believe that this is the first time that a strain of microsporidia has been isolated from the throat washings of a patient with microsporidiosis.

Inhibition of microsporidian spore germination

Microsporidia are obligate intracellular parasites that are increasingly recognized as significant causes of disease in AIDS patients. Gordon Leitch, Govinda Visvesvara and Qing He here describe the deployment of the microsporidian spore infection apparatus, the polar filament, and show how this may be a useful site for chemotherapeutic interdiction of the infections caused by these parasites.