Case Report: Molecular Diagnosis of Cystoisospora belli in a Severely Immunocompromised Patient with HIV and Kaposi Sarcoma.

Diarrhea in an immunocompromised patient has a broad infectious differential. Diagnosis is difficult despite advances in diagnostic modalities. We report a case of a 45-year-old Nigerian woman who immigrated to the United States 2 years ago. She presented to the hospital with gastrointestinal bleeding, newly diagnosed HIV, and disseminated Kaposi sarcoma. During hospitalization, the patient had an onset of watery diarrhea and high eosinophilia. Subsequent stool analysis using multi-parallel real-time quantitative polymerase chain reaction for 13 parasites was positive for Cystoisospora belli. The patient was treated with trimethoprim-sulfamethoxazole, but had relapsed disease when her antibiotics were stopped prematurely. After restarting trimethoprim-sulfamethoxazole, her diarrhea and eosinophilia improved, and she had undetectable Cystoisospora belli DNA on repeat stool quantitative polymerase chain reaction. This case highlights the importance of a thorough workup for diarrhea, including parasites, especially for immunocompromised patients. Antibiotic prophylaxis is recommended in patients with Cystoisospora belli and HIV/AIDS.

Epithelial Inclusions in Gallbladder Specimens Mimic Parasite Infection: Histologic and Molecular Examination of Reported Cystoisospora belli Infection in Gallbladders of Immunocompetent Patients.

Recent publications have described epithelial cytoplasmic vacuoles and inclusions incidentally noted within gallbladder epithelium and concluded that they represent coccidian parasite infection, in particular, Cystoisospora belli. We identified 8 gallbladder specimens from our institution in the past 3 years in which this diagnosis was suggested or in which similar epithelial alterations were prominent. Molecular analysis was performed on the 8 gallbladder specimens and on 3 positive control specimens: small bowel biopsies from acquired immunodeficiency syndrome patients with diarrhea. Polymerase chain reaction using primers designed to amplify an internal transcribed spacer (ITS2) in the C. belli ribosomal gene cluster was performed on the DNA samples. All 8 gallbladder specimens were negative for amplification, while a product consistent with C. belli was amplified from all 3 positive controls. Histologically, the gallbladder cytoplasmic inclusions stained diffusely positive for Grocott-Gomori's methenamine silver and Periodic acid-Schiff with diastase. In contrast, sections from a positive control small bowel biopsy demonstrated organisms that were negative for Grocott-Gomori's methenamine silver and showed a distinct capsular and punctate internal staining on Periodic acid-Schiff with diastase in various parasite forms. Together, the lack of molecular evidence of C. belli and the distinct morphologic and special staining patterns in these gallbladders compared with positive control small bowel suggest that these epithelial changes do not represent true C. belli infection. Our results suggest that gallbladders of immunocompetent patients may occasionally show epithelial changes that can morphologically mimic C. belli infection. Pathologists should be aware of this histologic variant to minimize unnecessary treatment, testing, and patient anxiety.

Infestación por Cryptosporidium Spp e Isospora Belli en preescolar inmunocompetente: a propósito de un caso / Infestation by Crytosporidium Spp and Isospora belli in immunocompetent preschool: a report of a case

El Cryptosporidium spp e Isospora belli son parásitos emergentes, que representan la cuarta causa de diarrea a nivel mundial, principalmente en niños y en pacientes inmunocomprometidos. Producen diarrea aguda o crónica dependiendo de la edad del paciente, estado nutricional e inmunológico asociado a factores sanitarios desfavorables. El diagnostico se realiza por visualización directa en heces con tinción de Zelh Neelsen modificado o Kinyou y biopsia intestinal con presencia de protozoos en las criptas y atrofia vellositaria de acuerdo al grado de infestación. Se reporta el caso de preescolar de 2 años de edad, eutrófico e inmunocompetente, perteneciente a estrato social bajo; con episodios de diarreas acuosas autolimitadas, dolor y distensión abdominal frecuentes. La biopsia intestinal revelo atrofia vellositaria e infestación simultanea por Cryptosporidium spp e Isospora belli corroborado por Tinción de Kinyou en heces; se descarto además Alergia Alimentaria, Enfermedad Celiaca e Inmunodeficiencias. El propósito de este caso clínico es alertar sobre la necesidad de incluir dentro del protocolo de estudio de diarrea crónica, la búsqueda de protozoarios formadores de esporas, mediante tinción especial en heces; un método no invasivo y sencillo, no solicitado en forma rutinaria.

Cryptosporidium spp and Isospora belli parasites are emerging that represent the fourth leading cause of diarrhea worldwide, mainly in children and in immunocompromised patients. Acute or chronic diarrhea occur depending on the patient's age, nutritional status and immunological factors associated with adverse health. The diagnosis is made by direct visualization in feces Neelsen stain Zelh Kinyou modified or intestinal biopsy and the presence of protozoa in the crypts and villous atrophy according to the degree of infestation. We report the case of preschool age 2, eutrophic immunocompetent, belonging to low socioeconomic levels, with self-limiting episodes of acute watery diarrhea, frequent abdominal pain and bloating. The intestinal biopsy revealed villous atrophy and simultaneous infestation by Cryptosporidium spp and Isospora belli Kinyou confirmed by staining in feces, while discarding also Food Allergy, Celiac disease and immunodeficiencies. The purpose of this case to alert about the need to include in the study protocol of chronic diarrhea, the search for spore-forming protozoa by special staining in feces, a noninvasive and simple method, not routinely requested.

Fat stores in a migratory bird: a reservoir of carotenoid pigments for times of need?

Carotenoids are well known for their immune-stimulant function in birds and other vertebrates. Moreover, they have potential antioxidant capacity, scavenging free radicals and protecting cell compartments from oxidation. Most essential carotenoids are fat soluble and could be stored for times of need especially in adipose tissues, built up by migratory birds as the main source of energy on long-distance flights. In an exclusive diet experiment, garden warblers (Sylvia borin) were fed ad libitum with an experimental diet, enriched with two different dose rates of carotenoids, or with control food, during the period of their first autumn migration. Plasma carotenoid content was measured via HPLC and chroma of plasma and fat examined with a spectrophotometer. Birds were infected with Isospora spp. and intensity of infection determined by oocyst counts 3 days post infection. Plasma lutein levels and chroma of subcutaneous fat stores were positively correlated and chroma values of these fat stores increased in the birds that got the higher dose rate, whereas they decreased significantly in the control group after infection with Isospora spp. Chroma of subcutaneous fat deposits in vivo and intensity of Isospora infection were negatively correlated. By measuring the chroma of fat deposits in vivo, we show that fat can be a reservoir for carotenoids. These colorful antioxidants are stored in the fat and taken from there in times of a higher demand, e.g. when mounting an immune response to parasites.

Faeces, FACS, and functional assays--preparation of Isospora suis oocyst antigen and representative controls for immunoassays.

SUMMARY: Highly purified antigen and appropriate controls are essential for antigen-specific immunoassays. In the case of Isospora suis, the causative agent of neonatal porcine coccidiosis, the only current source of antigen is oocysts isolated from faeces. The aim of this study was to develop a procedure for high-grade purification of I. suis oocysts from piglet faeces to obtain both antigen and representative controls suitable for in vitro re-stimulation of lymphocytes. This was achieved by use of filtration, density-gradient centrifugation and fluorescence-activated cell sorting (FACS). The feasibility for immunological studies was demonstrated with IFN-gamma ELISPOT assays after in vitro re-stimulation of lymphocytes from previously infected swine using the obtained antigen. The developed method allowed the production of highly purified antigen and representative controls from faeces with an oocyst recovery rate of 14%. Regarding the application of the obtained material it could be shown that lymphocytes from I. suis-infected pigs react in an antigen-specific manner in terms of an in vitro recall response by the production of IFN-gamma. This demonstrates the suitability of the developed method for the production of antigen and controls for sensitive immunological readout systems. Moreover, the detected specific IFN-gamma response encourages further functional studies on the cellular immune response to I. suis.

Changes in lymphocyte populations in suckling piglets during primary infections with Isospora suis.

Isospora suis, a common intestinal parasite of piglets, causes neonatal porcine coccidiosis, which results in reduced and uneven weaning weights and economic losses in pig production. Nevertheless, there are no detailed studies available on the immune response to I. suis. The aim of this study was to carry out phenotypical characterization of lymphocytes during primary infections on day 3 after birth. Infected and noninfected piglets were investigated between days 7 and 16 after birth. Lymphocytes from the blood, spleen and mesenteric lymph nodes (flow cytometry) and of the jejunal mucosa (immunohistochemistry) were analysed. A decrease in T cells, especially with the phenotype of resting T-helper cells, T-cell receptor-gammadelta-T cells, and regulatory T cells in the blood, spleen and mesenteric lymph nodes was noticeable. An increase in cells with the phenotype of natural killer cells in the spleen of infected animals was found, and the subset of TcR-gammadelta-T cells was strongly increased in the gut mucosa. Our findings suggest an accelerated migration of those cells into the gut. This study provides a strong indication for the involvement of adaptive and innate immune response mechanisms in the primary immune response to I. suis, especially of TcR-gammadelta-T cells as a linkage between innate and adaptive immunity.

Porcine coccidiosis--investigations on the cellular immune response against Isospora suis.

Porcine neonatal coccidiosis is caused by the protozoan Isospora suis and affects mainly piglets in the first three weeks of life. High morbidity with diarrhoea and reduced weight gain lead to economic losses, affecting pig-breeding worldwide. Infection causes damage of the mucosal surface in the jejunum and ileum and transient non-haemorrhagic diarrhoea. Secondary infections with other enteric pathogens may lead to increased mortality. Despite its economic and veterinary importance, the immunology of porcine isosporosis is still poorly understood. A striking feature of the infection is the rapidly increasing age resistance prohibiting the development of clinical disease in piglets older than 3-4 weeks irrespective of the immune status. It can be hypothesised that the development of the innate immune system in the first weeks of life and subsequently its interplay with the adaptive immune system is closely related to this phenomenon. Infections with I. suis induce migration of TcR-gammadelta(+) cells to the gut during primary infection and lead to induction of IFN-gamma production by TcR-gammadelta(+) cells and CD4(+) T-helper cells in blood and various lymphoid tissues. Like in other coccidial infections both innate as well as adaptive response mechanisms are activated during infection. They might be both not completely developed in the first weeks of life and therefore leaving a time frame for successful infection.

Cystoisospora canis Nemeséri, 1959 (syn. Isospora canis), infections in dogs: clinical signs, pathogenesis, and reproducible clinical disease in beagle dogs fed oocysts.

Canine intestinal coccidiosis is a cause of diarrhea in young dogs and dogs that are immunocompromised. Reports in the literature indicate that experimental reproduction of clinical coccidiosis with Cystoisospora canis (syn. Isospora canis) is difficult, and few studies have been done with C. canis. Experimental oral infections were attempted in 22, 6- to 8-wk-old female beagles with 5 x 10(4) (n = 2) or 1 x 10(5) (n = 20) sporulated C. canis oocysts. Diarrhea was observed in all inoculated dogs. Diarrhea began 2-3 days before oocyst excretion. Five of the 22 dogs were given an anticoccidial (sulfadimethoxine) because of their clinical signs. The mean prepatent period was 9.8 days (range, 9-11 days, n = 22 dogs), and the patent period was 8.9 days (range, 7-18 days, n = 20 dogs). Two dogs exhibiting clinical coccidiosis were examined at necropsy 10 days after infection. Developmental stages of C. canis were present in cells in the lamina propria throughout the entire small intestine in both dogs. Microscopic lesions observed in both of these dogs were villous atrophy, dilation of lacteals, and hyperplasia of lymph nodes in Peyer's patches. Results of bacterial and viral examinations of these 2 dogs were negative, indicating that intestinal coccidiosis was the cause of the diarrhea. Our study indicates that C. canis can be a primary cause of diarrhea in young dogs.

[Isosporiasis in an immunocompetent child: case report]. / Bagisiklik sistemi saglam bir çocukta isosporiasis: Olgu Sunumu.

A 12-year old boy was presented as a case report. He had been diagnosed with isosporiasis during a field study, conducted in a village of Manisa province, during an investigation of the incidence of intestinal parasites.

Porcine isosporosis: infection dynamics, pathophysiology and immunology of experimental infections.

Isospora suis, an intestinal protozoan parasite of swine, is the causative agent of neonatal coccidiosis, a disease with high morbidity in affected pig-breeding units and consequently of high economic importance. Infection leads to damage of the mucosal surface in the jejunum and ileum and to non-haemorrhagic diarrhoea. As a result, weight gain of piglets is reduced and secondary infections with other enteric pathogens may lead to increased mortality. Despite its economic and veterinary importance, host-parasite interactions are still poorly understood. To examine these interactions experimental infection models are established using outbred piglets infected with defined numbers of parasites on different days of life. This review discusses the life cycle of Isospora suis and the clinical and parasitological characteristics of porcine neonatal coccidiosis including pathology, and compare the different experimental infection models and the tools for studying Isospora suis in vitro. Moreover, it summarises findings about natural age resistance of pigs against infections with Isospora suis, our current knowledge about immune response to other coccidial infections, e.g. with Eimeria spp. in different hosts, and gives a short overview on peculiarities of the porcine immune system and its development in young animals which may play a role in porcine coccidiosis.

Atoxoplasmosis in tanagers.

Atoxoplasma spp. (extraintestinal Isospora spp.) are coccidian parasites that infect a variety of passerine species. Atoxoplasmosis has been difficult to diagnose using buffy coat and organ impression smear examinations or histopathologic examination of tissues at necropsy. The prevalence of this parasite was studied in the tanager collection of a zoological park after the death of several tanagers from confirmed and suspected infections. A polymerase chain reaction assay was used to test blood, feces, or tissue samples (or all) from 88 individuals representing 18 species. Twenty-three of 60 (38.3%) blood samples from clinically healthy birds tested positive for Atoxoplasma, and one of six fecal samples was positive. Nineteen of 32 (59.4%) tissue samples from deceased tanagers tested positive. A total of 57 other institutions were also queried regarding the presence of Atoxoplasma. The high number of Atoxoplasma-positive clinically healthy birds suggests that the parasite is prevalent subclinically within tanager collections, with young birds and stressed adults being the most likely to develop clinical disease. Thorough disinfection of enclosures, consideration of enclosure designs, and preventing fecal contamination of food and water are important methods of decreasing transmission. Selective breeding programs and regular screening of breeding pairs may be recommended to decrease transmission to susceptible offspring. Care should also be taken when housing tanagers in mixed species or in outdoor enclosures because the transmission risks between species have not been well established.

Role of acquired immunity and natural age resistance on course of Isospora suis coccidiosis in nursing piglets.

Thirty-two piglets from three litters were experimentally inoculated with 200000 sporulated oocysts of Isospora suis at 3 days of age and/or rechallenged at 19 days of age or primary inoculated at 19 days of age, to compare the role of acquired immunity and natural age resistance on the course of coccidiosis. Twelve piglets were not inoculated and served as a control. Following challenge, the signs of coccidiosis characterised by clinical symptoms, oocysts shedding and weekly weights were similar to those which occurred in piglets primary inoculated at 19 days of age. This comparison suggests that maturation of non-specific components of the immune system plays a more important role in the resistance of neonatal piglets to I. suis infection than specific immune mechanisms.

Demonstration of immunity against Isospora suis in swine.

Piglets naturally exposed or experimentally infected with Isospora suis oocysts were given challenge doses of oocysts to determine the extent of development of immune resistance. Piglets in both studies shed low numbers of, or no detectable oocysts, following challenge. Administration of methylprednisolone acetate failed to induce oocyst shedding in previously infected piglets. Piglets rechallenged with I. suis following steroid injections also failed to shed significant numbers of oocysts suggesting development of immunity to reinfection.