Biomedical subjects

G J Leitch

Publications and source records attributed to G J Leitch.

At least 19 recordsLinked to original sources

PubMed2002

Disseminated microsporidiosis in a renal transplant recipient.

Disseminated microsporidiosis is diagnosed uncommonly in patients not infected with human immunodeficiency virus (HIV). We present a case of disseminated microsporidiosis in a renal transplant recipient who was seronegative for HIV. Chromotrope-based stains were positive for microsporidia in urine, stools, sputum, and conjunctival scrapings. Electron microscopy, immunofluorescence, polymerase chain reaction, and cultures of renal tissue identified the organism as Encephalitozoon cuniculi. The patient was treated with oral albendazole and topical fumagillin with clinical improvement. In addition, she underwent a transplant nephrectomy and immunosuppressive therapy was withdrawn. Follow-up samples were negative for microsporidia. However, the patient developed central nervous system manifestations and died. An autopsy brain tissue specimen demonstrated E. cuniculi by immunofluorescent staining. Disseminated microsporidiosis must be considered in the differential diagnosis of multiorgan involvement in renal allograft recipients.

Animals↗

PubMed2001

Role of P glycoprotein in the course and treatment of Encephalitozoon microsporidiosis.

Encephalitozoon microsporidia are obligate intracellular protozoan parasites that proliferate and differentiate within a parasitophorous vacuole inside host cells that are usually epithelial in nature. Isolates of the three species of the Encephalitozoon microsporidia, E. cuniculi, E. hellem, and E. intestinalis, were obtained from AIDS patients and cultured in green monkey (E6) kidney cells. Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were used to assess the expression of Pgp and MRP respectively in uninfected and infected cells. Pgp, but not MRP, was detected immunocytochemically and by verapamil- and cyclosporin A-potentiated intracellular fluorescence in both host cells and parasite developing stages. When an in vitro infection assay was employed, verapamil and cyclosporin A acted as chemosensitizing agents for the antiparasitic drug albendazole. These observations suggest that inhibiting host cell and perhaps parasite MDR pumps may increase the efficacy of antiparasitic agents in these and other microsporidia species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

In vitro culture, ultrastructure, antigenic, and molecular characterization of Encephalitozoon cuniculi isolated from urine and sputum samples from a Spanish patient with AIDS.

In this report we describe the cultivation of two isolates of microsporidia, one from urine and the other from sputum samples from a Spanish AIDS patient. We identified them as Encephalitozoon cuniculi, type strain III (the dog genotype), based on ultrastructure, antigenic characteristics, PCR, and the sequence of the ribosomal DNA internal transcribed spacer region.

AIDS-Related Opportunistic Infections↗

PubMed2000

Local attenuation of systemically mediated splanchnic vasoconstriction during shock due to cholera.

Hypovolemic shock, most often due to hemorrhage, is typically associated with intense splanchnic vasoconstriction. This can be severe enough to impair the functional and structural integrity of the gastrointestinal tract. Paradoxically, with cholera the structure of the gastrointestinal tract is preserved, and the intestine continues to secrete fluid delivered to it in the circulating blood in spite of severe hypovolemic shock. This suggests that splanchnic blood flow is maintained at higher levels in hypovolemic shock due to cholera than in hypovolemic shock due to hemorrhage. Our hypothesis is that cholera toxin in the intestinal lumen activates local mechanisms that attenuate systemically mediated splanchnic vasoconstriction. Blood flow to an isolated ileal segment in situ in the anesthetized rabbit was measured continuously (ultrasound transit-time volume flow probe) for 5 to 6 h after instillation of cholera toxin into the isolated intestinal lumen. Norepinephrine was infused selectively into the mesenteric artery supplying the segment to elicit local responses uncomplicated by compensatory changes secondary to systemic effects of norepinephrine. Baseline vascular conductance increased gradually and became significantly greater in cholera toxin experiments than in vehicle experiments 5 h after treatment (P < 0.035). Animals treated with cholera toxin were less responsive to norepinephrine than vehicle treated animals were (P < 0.05) and became more so over time (P < 0.001). Our conclusion is that cholera toxin activates local mechanisms that attenuate systemically mediated splanchnic vasoconstriction, at least in part by reducing vascular responsiveness to a systemic vasoconstrictor, norepinephrine.

Animals↗

PubMed1999

Reactive nitrogen and oxygen species ameliorate experimental cryptosporidiosis in the neonatal BALB/c mouse model.

Four-day-old BALB/c mice were infected by the oral administration of 50,000 Cryptosporidium parvum oocysts, and the resulting infection was scored histologically and by counting colonic oocysts. Infection occurred in the ileum and proximal colon (but not duodenum and jejunum), peaked on days 14 to 18, and was cleared between days 24 and 30. Nitric oxide (NO) appeared to play a protective role in this model as evidenced by the facts that plasma nitrite and nitrate levels increased during the period of peak parasitosis; immunohistochemically detected inducible nitric oxide synthase (iNOS) was increased in the ileum and colon enterocytes of infected animals; the NOS inhibitor L-N-iminoethyl lysine or N-nitro-L-arginine methyl ester (L-NAME) decreased the elevated plasma nitrite and nitrate levels while exacerbating the infection and increasing oocyst shedding; administration of a NO donor, S-nitroso-N-penicillamine, reduced oocyst and infection scores; and neonatal iNOS knockout mice exhibited a slightly longer infection than control animals. The oral administration of oocysts to L-NAME-treated BALB/c mice, but not control animals, between 24 and 40 days old resulted in the fecal excretion of oocysts 1 week later. Administration of the antioxidant ascorbic acid also exacerbated the C. parvum infection, suggesting a protective role for reactive nitrogen and/or reactive oxygen compounds, while administration of the superoxide scavenger superoxide dismutase exacerbated the infection. Taken together these data suggest that both reactive nitrogen and reactive oxygen species play protective roles in experimental cryptosporidiosis.

Animals↗

PubMed1999

Detection by an immunofluorescence test of Encephalitozoon intestinalis spores in routinely formalin-fixed stool samples stored at room temperature.

Of the several microsporidia that infect humans, Enterocytozoon bieneusi is known to cause a gastrointestinal disease whereas Encephalitozoon intestinalis causes both a disseminated and an intestinal disease. Although several different staining techniques, including the chromotrope technique and its modifications, Uvitex 2B, and the quick-hot Gram-chromotrope procedure, detect microsporidian spores in fecal smears and other clinical samples, they do not identify the species of microsporidia. A need for an easily performed test therefore exists. We reevaluated 120 stool samples that had been found positive for microsporidia previously, using the quick-hot Gram-chromotrope technique, and segregated them into two groups on the basis of spore size. We also screened the smears by immunofluorescence microscopy, using a polyclonal rabbit anti-E. intestinalis serum at a dilution of 1:400. Spores in 29 (24.1%) of the 120 samples fluoresced brightly, indicating that they were E. intestinalis spores. No intense background or cross-reactivity with bacteria, yeasts, or other structures in the stool samples was seen. Additionally, the numbers of spores that fluoresced in seven of these samples were substantially smaller than the numbers of spores that were present in the stained smears, indicating that these samples were probably derived from patients with mixed infections of Enterocytozoon bieneusi and E. intestinalis. Because a 1:400 dilution of this serum does not react with culture-grown Encephalitozoon hellem, Encephalitozoon cuniculi, or Vittaforma corneae or with Enterocytozoon bieneusi spores in feces, we concluded that an immunofluorescence test using this serum is a good alternative for the specific identification of E. intestinalis infections.

AIDS-Related Opportunistic Infections↗

PubMed1998

Ultrastructure, immunofluorescence, western blot, and PCR analysis of eight isolates of Encephalitozoon (Septata) intestinalis established in culture from sputum and urine samples and duodenal aspirates of five patients with AIDS.

Microsporidia are ancient, intracellular, eukaryotic protozoan parasites that form spores and that lack mitochondria. Currently, as many as eight species included under six genera are known to infect humans, mostly patients with AIDS. Among these, Enterocytozoon bieneusi, the agent of gastrointestinal (GI) disease, is the most frequently identified microsporidian in clinical laboratories in the United States. Encephalitozoon (Septata) intestinalis, the agent that causes a disseminated infection including infection of the GI tract, is the second most frequently identified microsporidian parasite. In spite of this, not many isolates of E. intestinalis have been established in culture. We describe here the continuous cultivation of eight isolates of E. intestinalis obtained from different samples including the urine, sputum, and duodenal aspirate or biopsy specimens from five AIDS patients originating from California, Colorado, and Georgia. The specific identification was made on the bases of ultrastructural, antigenic, and PCR analyses.

AIDS-Related Opportunistic Infections↗

PubMed1998

Identification of the microsporidian Encephalitozoon hellem using immunoglobulin G monoclonal antibodies.

OBJECTIVE: Microsporidia isolated from clinical specimens so far have been identified to level of species by electron microscopy, indirect immunofluorescence (IIF), western blot (WB), and genetic analysis. Recent studies, however, indicate extensive serologic cross-reactions among microsporidian species involved in human disease. DESIGN AND SETTING: In this study, we used IIF and WB techniques to evaluate the reactivity of six different immunoglobulin G monoclonal antibodies (MAbs) raised against Encephalitozoon hellem with six isolates of E hellem that originated from patients with acquired immunodeficiency syndrome. A rabbit isolate of Encephalitozoon cuniculi, and an isolate of Encephalitozoon intestinalis, which was established in cultures from the urine of a patient with acquired immunodeficiency syndrome were also used for comparison. RESULTS: Five of the six antibodies, when analyzed by both IIF and WB assays, specifically identified six isolates of E hellem originating from three patients with acquired immunodeficiency syndrome. The sixth MAb, however, reacted with all of the E hellem isolates in the WB assay, but failed to react with them in the IIF assay. Using the IIF test, five of the six MAbs failed to react with E cuniculi and E intestinalis, even at a dilution of 1:50. The MAbs also did not react in the IIF test with Enterocytozoon bieneusi, Giardia, and Cryptosporidium. These MAbs did react with E cuniculi and E intestinalis in the WB assay, but the banding patterns were very different from those of E hellem, thus facilitating the identification of E hellem from the other microsporidia. The MAbs also reacted, in the IIF test, with E hellem spores in formalin-fixed tissue sections that were heated in a microwave oven. CONCLUSIONS: Identification of microsporidian agents to the species level is important. Since certain therapeutic agents (eg, fumagillin, albendazole) are efficacious in treating E hellem infections of the cornea, as well as urogenital and respiratory infections caused by E hellem, a quick and definitive identification of the organism is important so that successful therapy may be instituted. An IIF test using the MAbs described here would therefore be invaluable in the quick identification of this parasite.

Animals↗

PubMed1997

Use of a fluorescent probe to assess the activities of candidate agents against intracellular forms of Encephalitozoon microsporidia.

Microsporidia are obligate intracellular protozoan parasites. Three species of the genus Encephalitozoon are among the microsporidia that infect immunodeficient humans. These species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis, all develop in a parasitophorous vacuole within a host cell. The present study describes a method that uses the fluorescent probe calcein and confocal microscopy to detect drug-induced effects in Encephalitozoon-infected green monkey kidney cells. The effects were as follows: (i) changes in parasite organization within the parasitophorous vacuole; (ii) swelling and gross morphological changes of parasite developing stages in situ; (iii) killing of developing parasite stages in situ, detected by their uptake of the fluorescent probe; and (iv) reduction in the viability of the host cell population, assessed by the loss of the probe. Verapamil and itraconazole were used to increase the vital dye loading by both uninfected and infected cells. Agents with known antimicrosporidial activity, albendazole and fumagillin, caused all three types of parasite changes at concentrations that had no detectable effect on host cell viability. The effective doses of albendazole and fumagillin that caused swelling and disorganization of parasite developing stages were 5 x 10(-7) and 10(-6) M respectively. Killing of developing stages was detected at 10-fold-higher concentrations for these agents and at 10(-5) M for metronidazole. This method can be used to screen candidate antimicrosporidial agents in infected cultured cells.

Albendazole↗

PubMed1997

Western blot and immunofluorescence analysis of a human isolate of Encephalitozoon cuniculi established in culture from the urine of a patient with AIDS.

Microsporidia spores, identified as Encephalitozoon cuniculi (CDC: V282), were isolated from the urine of a patient with acquired immunodeficiency syndrome and disseminated microsporidiosis, established in continuous culture on monkey kidney cells (E6), and antiserum was produced in rabbits. Immunoblot studies that used the patient serum and the rabbit sera against CDC:V282, Encephalitozoon hellem (CDC:0291:V213), and Encephalitozoon intestinalis (CDC:V297) revealed that CDC:V282 and the rabbit isolate of E. cuniculi (ECLD) reacted intensely with the patient's serum and the rabbit anti-CDC:V282, producing a number of bands ranging from 200 to 15 kDa. By contrast, the heterologous antigens (CDC:0291:V213 and CDCV297) reacted minimally. Both CDC:V282 and ECLD isolates of E. cuniculi reacted minimally with the rabbit anti-E. hellem and the rabbit anti-E. intestinalis sera. In the immunofluorescence test, performed on the lung biopsy section of the patient, the rabbit anti-CDC:V282 serum reacted extensively with the spores in the tissue section and produced bright apple green fluorescence. These studies demonstrated that the human (CDC:282) and the rabbit (ECLD) isolates of E. cuniculi were similar in their antigenic profiles but differed considerably from E. hellem and E. intestinalis, and that the patient's serum reacted specifically, strongly, and with equal intensity, with the 2 isolates of E. cuniculi.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Effects of nifedipine, metronidazole, and nitric oxide donors on spore germination and cell culture infection of the microsporidia Encephalitozoon hellem and Encephalitozoon intestinalis.

Two species of microsporidia, Encephalitozoon hellem and Encephalitozoon intestinalis, were isolated from AIDS patients and cultured in green monkey kidney cells. A spore germination assay and a cultured-cell infection assay were used to test the efficacy of candidate antiparasitic agents. The calcium channel blocker nifedipine, metronidazole, and two nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, were tested in the two assays. Nifedipine (10(-8) M) significantly inhibited E. hellem spore germination in three of four germination media. Metronidazole (10(-5) M) inhibited germination weakly and significantly inhibited E. intestinalis germination in a single germination medium. The inhibitory effect of nifedipine and metronidazole used together was greater than the sum of the effects of the drugs used alone in all E. hellem germination assays. The NO donors also inhibited spore germination. The inhibitory effect of nifedipine and metronidazole could be reversed by washing the spores, while that of the NO donors was not reversible. In early cultured-cell infections, both nifedipine (10(-8) M) and metronidazole (10(-5) M) significantly reduced the number of cells being infected. As the infection spread, these agents were less effective. Some inhibition of the spread of the infection was also demonstrated with the NO donors at a concentration (10(-5) M) not obviously toxic to the cultured cells. These data suggest that combination drug therapy targeting spore germination and intracellular parasite development is promising.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Adenovirus masquerading as microsporidia.

Enterocytozoon bieneusi is a microsporidian that causes a severe, debilitating, chronic diarrhea in some patients with the acquired immunodeficiency syndrome. Specific diagnosis of E. bieneusi currently requires an invasive biopsy procedure and time-consuming preparation of specimens for electron microscopy. Our attempts to establish an in vitro culture system using mammalian cell cultures inoculated with duodenal aspirates, biopsy, or both, from 2 infected patients resulted in inadvertent coculture of an adenovirus and E. bieneusi. The adenovirus-infected cells deceptively appeared to contain spores of microsporidia based on light microscopic examination. Transmission electron microscopy revealed only a few microsporidia, but numerous cells infected with an adenovirus that was subsequently identified as adenovirus type 8. We believe that adenovirus infections prevented the cultured cells from supporting the proliferation of E. bieneusi and ultimately destroyed the cell cultures.

AIDS-Related Opportunistic Infections↗

PubMed1996

Pathology of microsporidiosis: emerging parasitic infections in patients with acquired immunodeficiency syndrome.

OBJECTIVE: Microsporidiosis is a group of rapidly emerging protozoan infections that have thus far been reported predominantly from severely immunosuppressed persons with the acquired immunodeficiency syndrome (AIDS). The four genera that have been identified in AIDS patients (Enterocytozoon, Encephalitozoon, Septata, and Pleistophora) are an increasingly common source of both localized and disseminated infections. However, the clinical and pathologic features of these agents are being described with such rapidity that many pathologists are unaware of the histologic, immunologic, and molecular methods for diagnosing these infections. This article summarizes the clinical and morphologic spectrum of the microsporidian species that infect patients with AIDS. Additionally, the role of ultrastructural, immunologic, tissue culture, and molecular techniques for the diagnosis of microsporidian infections are discussed. DATA SOURCES: Clinical and pathologic findings were obtained from patients with AIDS who were evaluated for microsporidian infections at the Grady Memorial Hospital in Atlanta. Selected laboratory studies were performed at the Division of Parasitic Diseases of the Centers for Disease Control and Prevention and at the Department of Physiology at Morehouse University. Additionally, some cases were sent for consultation to the Infectious Disease Pathology service at Emory University. These data were combined with the published studies of microsporidian infection from the medical literature. DATA SYNTHESIS: The pathologic appearance of microsporidian infections in each major organ system (ocular, respiratory, genitourinary, gastrointestinal) is illustrated using routine and special histochemistry and immunofluorescence. The differential diagnostic features of the four genera of microsporidia infecting AIDS patients are illustrated using transmission and scanning electron micrographs from biopsy, autopsy, and tissue culture materials. Cytologic evaluation of body tissues is emphasized as a sensitive method for microsporidian diagnosis. CONCLUSIONS: Microsporidian infections can be expected to remain an increasingly important cause of morbidity and mortality in patients with AIDS. It is important that pathologists and microbiologists become acquainted with the clinicopathologic spectrum of these emerging protozoal infections, ensuring timely diagnosis and subsequent treatment.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Polymerase chain reaction and culture confirmation of disseminated Encephalitozoon cuniculi in a patient with AIDS: successful therapy with albendazole.

Infections due to microsporidia are being recognized increasingly, especially in AIDS patients. A patient with disseminated microsporidiosis with advanced renal failure due to Encephalitozoon cuniculi (confirmed by culture and polymerase chain reaction [PCR]) is described. The organism from urine and sputum was characterized by culture, Weber's chromotrope-based staining, transmission electron microscopy, and indirect immunofluorescence (IIF) tests. PCR was done on DNA extracted from the infected cell cultures. Treatment with albendazole resulted in improvement in serum creatinine levels, complete disappearance of spores from sputum, a negative urine culture, and a 3-log decline in the number of spores in the urine, as evidenced by chromotrope-based staining. IIF and PCR were used to confirm E. cuniculi as the etiologic agent. Our findings indicate that disseminated microsporidiosis with renal failure in AIDS is treatable.

AIDS-Related Opportunistic Infections↗

PubMed1995

In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS.

Microsporidian spores were identified, on the basis of Weber's staining, in urine, stool, nasal, and saliva samples of an AIDS patient with diarrhea, hematuria, dysuria, and dementia. Urine and stool samples contained numerous spores, whereas few spores were seen in the nasal and saliva samples. Spores were concentrated from urine samples and inoculated into monkey kidney cell (E6) monolayers. After 6 to 8 weeks of culture, infected E6 cells filled with spores as well as spores free in the culture supernatants were seen daily. Transmission electron microscopy revealed that all stages of the parasite (CDC:V297) developed within septated, honeycomb-shaped parasitophorous vacuoles. Indirect immunofluorescence and immunoblotting studies using rabbit anti-Encephalitozoon cuniculi, anti-Encephalitozoon hellem, and anti-CDC:V297 sera revealed that CDC:V297 reacted intensely with the homologous serum but minimally with the heterologous sera. DNA isolated from CDC:V297, when PCR amplified with E. hellem and E. cuniculi primers, did not produce the diagnostic bands of approximately 547 and approximately 549 bp characteristic of E. hellem and E. cuniculi, respectively. On the basis of these studies, we concluded that CDC:V297 fits the description of Septata intestinalis (A. Cali, D. P. Kotler, and J. M. Orenstein, J. Eukaryot, Microbiol. 40:101-112, 1993).

AIDS-Related Opportunistic Infections↗

PubMed1994

Putative anticryptosporidial agents tested with an immunodeficient mouse model.

Lasalocid, sinefungin, and dehydroepiandrosterone were tested for anticryptosporidial activity with an immunodeficient mouse model at doses that have been reported effective when tested with immunosuppressed rodent models. Small but significant reductions in oocyst excretion were only observed under some conditions with lasalocid and dehydroepiandrosterone, but sinefungin had no effect.

Adenosine↗

PubMed1994

Arginine-derived nitric oxide reduces fecal oocyst shedding in nude mice infected with Cryptosporidium parvum.

Dietary L-arginine (4%) significantly reduced fecal oocyst shedding in athymic nude mice chronically infected with Cryptosporidium parvum. This effect appeared to be due to an increase in host nitric oxide (NO) production as it was not observed in arginine-supplemented animals administered the NO synthase inhibitor, N-nitro-L-arginine methyl ester. N-Nitro-L-arginine methyl ester alone significantly increased fecal oocyst shedding in chronically infected animals. In in vitro assays, oocyst excystation and sporozoite viability were significantly reduced by the NO donors sodium nitroprusside and S-nitroso-L-acetyl penicillamine in a concentration-dependent manner. These data suggest that arginine-derived NO may reduce the parasite load in experimental cryptosporidiosis.

Amino Acid Oxidoreductases↗

PubMed1994

Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection.

Microsporidia are primitive, spore-forming, mitochondria-lacking, eukaryotic protozoa that are obligate intracellular parasites. They are known to parasitize almost every group of animals including humans. Recently, microsporidia have increasingly been found to infect patients with AIDS. Five genera (Encephalitozoon, Enterocytozoon, Nosema, Septata, and Pleistophora) of microsporidia are known to infect humans. Enterocytozoon organisms cause gastrointestinal disease in a majority of AIDS patients with microsporidiosis. However, a smaller, but an expanding, number of patients with AIDS are being diagnosed with ocular and disseminated infection with Encephalitozoon hellem. Although microsporidial spores can be identified in clinical samples by a staining technique such as one with Weber's chromotrope stain, identification to the species level is dependent on cumbersome and time-consuming electron microscopy. We have recently isolated and established in continuous culture several strains of E. hellem from urine, bronchoalveolar lavage, and sputum samples from AIDS patients with disseminated microsporidiosis. We developed polyclonal and monoclonal antibodies and PCR primers to a strain of E. hellem that can be used successfully to identify E. hellem from other species of microsporidia either in clinical specimens or in cultures established from clinical specimens. Since patients infected with Encephalitozoon spp. are known to respond favorably to albendazole, identification of the parasite to the species level would be invaluable in the treatment of disseminated microsporidiosis.

Acquired Immunodeficiency Syndrome↗