Cystoisospora belli, liver disease and hypothesis on the life cycle.

Cystoisospora belli causes chronic diarrhoea, acalculous cholecystitis, cholangiopathy and disseminated cystoisosporosis in patients with AIDS. Clinical manifestations and histological stages during C. belli infection in a patient with AIDS and liver disease were described. It was possible to identify sporozoite-like structures in the villus epithelium of the duodenum, close to the vascularization that underlies the basal membrane and unizoite tissue cysts near to the vascularization in the lamina propria. Unizoite tissue cysts were found inside of sinusoids in the liver communicating with the central vein and with a bile canaliculus and portal spaces. Based on these findings a hypothesis on C. belli life cycle could consider that the route of migration of unizoite tissue cysts up the liver is via the portal blood. The unizoite tissue cysts located in hepatic portal vein could migrated via sinusoid to central vein and general circulation through the venous system. The unizoite tissue cysts could also return via bile canaliculus to bile duct to portal triad. This hypothesis allows to understand the presence of unizoite stages in blood, the pathway by which the bile ducts become infected and unizoites in the liver being able to behave like hypnozoites that favour relapses and treatment failures.

Microscopic and PCR-based detection of microsporidia spores in human stool samples.

Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrhea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

First detection of Cryptosporidium DNA in blood and cerebrospinal fluid of HIV-infected patients.

Human cryptosporidiosis is an intestinal infection caused by different species belonging to the genus Cryptosporidium in both immunocompetent and immunocompromised individuals. The life cycle of Cryptosporidium sp. when affecting the digestive system is well known but the infection of other organs is less studied. Molecular methods are necessary for species and subtypes identification. The goal of this work is to propose a new approach that contributes to the diagnosis of the extra-intestinal dissemination process of Cryptosporidium infection. Cryptosporidium sp. was detected in stool and biopsy samples of two HIV-infected patients. DNA was extracted from feces, biopsy specimens, blood, and cerebrospinal fluid (CSF). All samples were analyzed by nested PCR-RFLP of the 18S rDNA, real-time PCR, and gp60 subtyping. Cryptosporidium DNA was detected in stool and tissue samples and it was also present in blood and CSF samples. Both cases were characterized as Cryptosporidium hominis subtype IeA11G3T3. This is the first report that demonstrates the presence of Cryptosporidium DNA in blood and CSF of HIV-infected patients.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina.

The identification and characterisation of Cryptosporidium genotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity of Cryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays for Cryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.

Molecular identification of protozoa causing AIDS-associated cholangiopathy in Buenos Aires, Argentina.

BACKGROUND: Several species of microsporidia and coccidia are protozoa parasites responsible for cholan-giopathy disease in patients infected with human immunodeficiency virus (HIV). The goals of this work were to identift opportunistic protozoa by molecular methods and describe the clinical manifestations at the gastrointestinal tract and the biliary system in patients with AIDS-associated cholangiopathy from Buenos Aires, Argentina. MATERIAL AND METHODS: This study included 11 adult HIV-infected individuals with diagnosis ofAIDS- associated cholangiopathy. An upper gastrointestinal endoscopy with biopsy specimen collection and a stool analysis for parasites were performed on each patient. The ultrasound analysis revealed bile ducts compromise. An endoscopic retrograde cholangiopancreatography and a magnetic resonance cholangiography were carried out. The identification to the species level was performed on biopsy specimens by molecular methods. RESULTS: Microorganisms were identified in 10 cases. The diagnosis in patients with sclerosing cholangitis was cryptosporidiosis in 3 cases, cystoisosporosis in 1 and microsporidiosis in 1. In patients with sclerosing cholangitis and papillary stenosis the diagnosis was microsporidiosis in 2 cases, cryptosporidiosis in 2 and cryptosporidiosis associated with microsporidiosis in 1. In 3 cases with cryptosporidiosis the species was Cryptosporidium hominis, 1 of them was associated with Enterocytozoon bieneusi, and the other 2 were coinfected with Cryptosporidium parvum. In the 4 cases with microsporidiosis the species was Enterocytozoon bieneusi. CONCLUSIONS: These results suggest that molecular methods may be useful tools to identify emerging protozoa in patients with AIDS-associated cholangiopathy.

Diagnostic Features of Blastocystis Life Cycle Forms in the Small Intestine in an HIV-Infected Patient.

PURPOSE: Blastocystis spp. are parasites of the intestinal tract found in many hosts including humans. This pathogen is commonly found in immunocompetent in asymptomatic individuals and in patients with gastrointestinal and extra-intestinal symptoms. Recently, it has been implicated as an important cause of diarrheal illness in immunocompromised individuals, including HIV-infected patients. At least six life cycle stages have been described in faeces and cultures, namely vacuolar, granular, multi-vacuolar, avacuolar, ameboid and cyst forms. The aim of the present study was to describe the histological findings of Blastocystis infection in an adult HIV-infected patient with gastrointestinal symptoms. METHODS: Parasitological techniques and PCR were applied to stool samples. Histological analysis was performed on duodenal biopsy specimens. RESULTS: Standard parasitological methods revealed vacuolar, granular, cyst and multi-vacuolar forms of Blastocystis in faecal samples with the presence of Blastocystis DNA being confirmed by PCR. DNA sequencing revealed Blastocystis subtype ST1. Histological findings in duodenal samples showed an inflammatory infiltrate with plasma cells and lymphocytes. We identified cyst, granular, ameboid and multi-vacuolar forms in the lumen. CONCLUSION: To our knowledge, there are no previous peer review reports describing these four different forms of Blastocystis in histological sections from the lumen and the brush border of the enterocytes.

Molecular characterization of Cystoisospora belli and unizoite tissue cyst in patients with Acquired Immunodeficiency Syndrome.

Cystoisospora belli is a coccidian protozoan that can cause chronic diarrhoea, acalculous cholecystitis and cholangiopathy in AIDS patients. We applied molecular methods to identify Cystoisospora at species level in AIDS patients presenting with and without the presence of unizoites in lamina propria. Coprological and histological analyses were performed in stool and/or biopsy samples from 8 Cystoisospora-infected patients. DNA from the same samples was used to amplify 2 fragments of the SSU-rRNA gene and the ITS-1 region. Sequencing of the resulting amplicons identified C. belli infections in all cases, independent of the presence or absence of unizoite tissue cysts. Further work should be considered in order to find molecular targets related to strain variations in C. belli.

Use of the PCR in a Combined Methodological Approach for the Study of Human Fascioliasis in an Endemic Area.

PURPOSE: Fascioliasis is a worldwide distributed trematodiasis considered a neglected disease. Diagnosis in humans has been traditionally based on parasitological and immunological techniques. Recently we reported the use of the PCR in stool samples for the individual diagnosis. The purpose of this study was to evaluate human fascioliasis by a combination of diagnostic methods in an area where the disease is highly endemic in animals. METHODS: We studied all the inhabitants (N = 240) of Tatón village, Argentina, by Fasciola hepatica rproCL1-ELISA. Among them, we continued the study with 13 cases that had at least two positive serological tests, who performed a questionnaire, physical examination, abdominal ultrasonography, and collection of blood and faeces. Blood/serum samples were used for Fh rproCL1-ELISA and liver function tests. Faeces were used for parasitological analysis and PCR of a repetitive fragment of Fasciola sp. RESULTS: Among the 13 patients, 9 presented symptoms of biliary colic. All patients repeated positive serology. F. hepatica eggs were not detected. PCR was positive in 11 cases. CONCLUSION: This is the first report employing an approach based on the combination of methods for the evaluation of human fascioliasis in an endemic area, which includes molecular tools with a high value in detecting low infections.

[Identification of Cryptosporidium hominis in a patient with sclerosing cholangitis and AIDS]. / Identificación de Cryptosporidium hominis en un paciente con colangitis esclerosante y SIDA.

Cryptosporidium hominis (C hominis) is the most common protozoan parasite recognized in human patients with AIDS. We report the clinical features of a patient with chronic diarrhea and AIDS-related sclerosing cholangitis. The imaging studies with ultrasonography and endoscopic retrograde cholangiopancreatography disclosed intrahepatic and extrahepatic bile duct changes identical to those seen in sclerosing cholangitis. C hominis was detected in the duodenum and peri-papillary duodenum by means of light microscopy and confirmed by nested polymerase chain reaction (PCR) amplification from fresh biopsy specimens followed by restriction length polymorphism analysis. Chominis infection should be suspected in our country in patients with advanced immunodeficiency and AIDS-related sclerosing cholangitis.

Multimethodological Approach to Gastrointestinal Microsporidiosis in HIV-Infected Patients.

PURPOSE: Microsporidiosis is an opportunistic infection that produces chronic diarrhoea and cholangiopathy in patients with AIDS, mainly caused by two species of microsporidia, Enterocytozoon bieneusi and Encephalitozon intestinalis. The aim of this work was to develop an integral system for the diagnosis of microsporidiosis of the intestine and biliary tract in HIV-infected patients, comprising microscopic and molecular techniques. METHODS: The study population comprised 143 adult patients of both sexes with diagnosis of HIV infection, with chronic diarrhoea, and with or without HIV-associated cholangiopathy. Stool studies for microsporidia identification of spores were performed on each patient. A video esofagogastroduodenoscopy with biopsy collection was also carried out for routine histology and semi-thin sections stained with Azure II. Species identification was carried out by transmission electron microscopy and/or polymerase chain reaction for the species E. bieneusi and E. intestinalis. RESULTS: Out of the 143 patients a total of 12.6% (n = 18) were infected with microsporidia. Microsporidia species identified in most cases was E. bieneusi (16/18 cases), followed by E. intestinalis (4/18), all of these last ones in coinfection with E. bieneusi. CONCLUSIONS: Clinical, imaging, microscopic and molecular analyses, when applied in a systematic and integrated approach, allow diagnosis and identification of microsporidia at species level in AIDS patients with chronic diarrhoea, and with or without HIV-associated cholangiopathy.

Fatal canine encephalitozoonosis in Latin America, first report.

Encephalitozoon cuniculi is an obligate, intracellular microsporidian organism capable of establish infection in a wide variety of animals. In carnivores it may cause a sporadic, severe disease in the first few months of life, which usually culminates with the death of the animal. The objective of this study was to report a natural fatal case of encephalitozoonosis in a puppy from Argentina. Clinical signs included reduced appetite, depression, vocalizing, weight loss, weakness, convulsions and recumbency. No significant gross lesions were noticed at necropsy. Microscopically, severe, diffuse, lymphocytic encephalitis was seen. Large cytoplasmic vacuoles containing spores, morphologically compatible with E. cuniculi, were present within endothelial cells of brain and kidney, in renal tubular epithelium and hepatocytes. Encephalitozoon cuniculi DNA was detected by PCR in the kidney. Antibody titers to E. cuniculi in serum from the surviving puppies and the dam were ≥1:200. This report contributes to our understanding of neurologic disease in puppies. Encephalitozoonosis should be considered as a differential diagnosis in cases of fatal encephalitis in puppies.

First genetic characterization of Fasciola hepatica in Argentina by nuclear and mitochondrial gene markers.

Fasciola hepatica is a trematode showing genetic variation among isolates from different regions of the world. The objective of this work was to characterize for the first time F. hepatica isolates circulating in different regions of Argentina. Twenty-two adult flukes were collected from naturally infected bovine livers in different areas from Argentina and used for DNA extraction. We carried out PCR amplification and sequence analysis of the ribosomal internal transcribed spacer 1 (ITS1), mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunits 4 and 5 (nad4 and nad5) and mitochondrial cytochrome c oxidase subunit I (cox1) genes as genetic markers. Phylogenies were reconstructed using maximum parsimony algorithm. A total of 6 haplotypes were found for cox1, 4 haplotypes for nad4 and 3 haplotypes for nad5. The sequenced ITS1 fragment was identical in all samples. The analyzed cox1 gene fragment is the most variable marker and is recommended for future analyses. No geographic association was found in the Argentinean samples.

PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails.

Fasciolosis, caused by the trematode Fasciola hepatica, is a zoonosis of economic importance in livestock that is emerging as a chronic disease in humans. The intermediate hosts are lymnaeid snails, in which diagnosis of infection is traditionally based on cercarial shedding, tissue sectioning and crushing. We developed a PCR assay for the sensitive and specific detection of F. hepatica in field-collected Lymnaea sp. snails. A primer pair was designed to amplify a 405 bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica. The PCR assay showed a limit of detection of 10 pg of genomic F. hepatica DNA. No cross-reactions were observed with samples from other related trematode species or from the snail hosts Lymnaea columella and Lymnaea viatrix. DNA sequencing of the amplicon showed 100% homology with F. hepatica, and 75-89% homology with other trematodes on regions that did not include the entire set of primers. Two samples from Argentina were analysed. For snails in sample 1 (n = 240), identified as L. columella, the infection rate was 17.5 and 51.3% by direct examination and PCR, respectively. For snails in sample 2 (n = 34), identified as L. viatrix, the infection rate was 2.9 and 61.8% by direct examination and PCR, respectively. Differences in infection rates between these diagnosis methods were significant for both samples. Our PCR technique showed to be effective for detecting specific F. hepatica infections of low intensity in the intermediate host, and hence it could be used to study the epidemiological situation in a given area, as well as to assess host suitability for the parasite.

Pulmonary toxoplasmosis in human immunodeficiency virus-infected patients in the era of antiretroviral therapy.

Toxoplasmosis is a severe opportunistic infection in patients infected with the human immunodeficiency virus (HIV). The lung is a major site of infection after the central nervous system. In this report we described two cases of pneumonia due to Toxoplasma gondii infection in HIV patients with antiretroviral therapy. Clinical and radiological abnormalities are not specific. Pulmonary toxoplasmosis should be considered in HIV-infected patients with late stage of HIV, CD4 count less than 100 cells/µl and a poor adherence to HAART.

Human fascioliasis endemic areas in Argentina: multigene characterisation of the lymnaeid vectors and climatic-environmental assessment of the transmission pattern.

BACKGROUND: In South America, fascioliasis stands out due to the human endemic areas in many countries. In Argentina, human endemic areas have recently been detected. Lymnaeid vectors were studied in two human endemic localities of Catamarca province: Locality A beside Taton and Rio Grande villages; Locality B close to Recreo town. METHODS: Lymnaeids were characterised by the complete sequences of rDNA ITS-2 and ITS-1 and fragments of the mtDNA 16S and cox1. Shell morphometry was studied with the aid of a computer image analysis system. Climate analyses were made by nearest neighbour interpolation from FAO data. Koeppen & Budyko climate classifications were used. De Martonne aridity index and Gorczynski continentality index were obtained. Lymnaeid distribution was assessed in environmental studies. RESULTS: DNA sequences demonstrated the presence of Lymnaea neotropica and L. viator in Locality A and of L. neotropica in Locality B. Two and four new haplotypes were found in L. neotropica and L. viator, respectively. For interspecific differentiation, ITS-1 and 16S showed the highest and lowest resolution, respectively. For intraspecific analyses, cox1 was the best marker and ITS-1 the worst. Shell intraspecific variability overlapped in both species, except maximum length which was greater in L. viator. The desertic-arid conditions surrounding Locality A, the semiaridity-aridity surrounding Locality B, and the very low yearly precipitation in both localities, are very different from the typical fascioliasis transmission foci. Lymnaeids are confined to lateral river side floodings and small man-made irrigation systems. Water availability only depends on the rivers flowing from neighbouring mountains. All disease transmission factors are concentrated in small areas where humans and animals go for water supply, vegetable cultures and livestock farming. CONCLUSIONS: The unusually high number of DNA haplotypes and the extreme climate unsuitable for F. hepatica and lymnaeid development, demonstrate that the transmission foci are isolated. Seasonal transmission may depend on the timely overlap of appropriate temperature and river water availability. Lymnaeids and F. hepatica have probably reached these localities by livestock introduction. DNA differences regarding other populations of L. neotropica and L. viator in Argentina suggest an introduction independent from the spreading movements which allowed these two lymnaeids to expand throughout the country.

First report of Cystoisospora belli parasitemia in a patient with acquired immunodeficiency syndrome.

Cystoisospora belli in patients with the acquired immunodeficiency syndrome (AIDS) has been described as cause of chronic diarrhea and disseminated cystoisosporosis. Diagnosis of intestinal cystoisosporosis can be achieved at the tissue level in the villus epithelium of the small bowel. Disseminated cystoisosporosis is diagnosed by microscopy identification of unizoite tissue cysts in the lamina propria of the intestine. We report a case of disseminated cystoisosporosis in a human immunodeficiency virus (HIV)-infected patient with detection of parasitemia. We studied a 39-year old patient with AIDS and chronic diarrhea by analysis of stool and duodenal biopsy samples. Blood samples were also collected and examined by light microscopy and molecular techniques for C. belli DNA detection. The unizoite tissue cyst stages were present in the lamina propria, with unsporulated oocysts in feces. Zoites were present in blood smears and DNA of C. belli was detected in blood samples. Our study identified a new stage in the life cycle of C. belli. Detection of parasitemia is a novel and noninvasive tool for diagnosis of disseminated cystoisosporosis.

Fasciola hepatica infection in humans: overcoming problems for the diagnosis.

Fascioliasis is a zoonosis actually considered as a foodborne trematode disease priority by the World Health Organization. Our study presents three cases of F. hepatica infection diagnosed by direct, indirect and/or imaging diagnostic techniques, showing the need of the combined use of them. In order to overcome some difficulties of the presently available methods we show for the first time the application of molecular tools to improve human fascioliasis diagnosis by employing a PCR protocol based on a repetitive element as target sequence. In conclusion, diagnosis of human fascioliasis has to be carried out by the combination of diagnostic techniques that allow the detection of infection in different disease phases, different epidemiological situations and known/new transmission patterns in the actual scenario.

Microscopio and PCR-based detection of microsporidia spores in human stool samples

Abstract Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

Resumen Los microsporidios son hongos intracelulares obligados con una notable capacidad para infectar una amplia gama de hospedadores invertebrados y vertebrados. Enterocytozoon bieneusi es el microsporidio más frecuentemente reportado en todo el mundo, principalmente tricrómicaasociado con diarrea crónica y síndrome debilitante en pacientes con sida. Las técnicas dedetección basadas en microscopía y PCR son útiles para el diagnóstico y la identificación deespecies y genotipos, pero estos métodos deben estar estandarizados en cada laboratorio.En este estudio evaluamos técnicas de microscopía y PCR anidada, con secuenciación de losproductos, para detectar E. bieneusi en muestras de heces humanas. Estas técnicas, usadas con-juntamente, podrían ser útiles para su aplicación en el diagnóstico de microsporidiosis intestinaly para realizar estudios epidemiológicos de esta afección en Argentina.

[A focus study from a case of human fascioliasis in Neuquén]. / Estudio del foco en un caso de fasciolosis humana en Neuquén.

An epidemiological focal study was performed in Loncopué, Neuquén, Argentina, in November 2002 to detect the origin of the infection in a human case of fascioliasis confirmed by an indirect-ELISA test, six months before the study. Thirty five individual fecal samples were taken from domestic livestock, and watercress plants and snails were collected from the irrigation ditches connected to a main canal in the surroundings of the patient's house. A new blood sample was taken from the already recovered patient. The patient was still seropositive to Fasciola hepatica antigens. No metacercariae were found in the 222 watercress leaves checked. All the snails collected (n=130) were identified as Lymnaea viatrix and two out of 101 (2%) were infected with F. hepatica larvae. Coprological analysis showed F. hepatica eggs in 100% of goats (10/10), 82% of sheep (9/11) and 86% of bovines (6/7). The number of eggs per gram shed by positive goats (median=20.7, Q1=6.2, Q3=34.5) and sheep (4, 1.8, 13) was significantly higher than in cows (0.3, 0.3, 1.7) (p < 0.01). Local veterinary control programs were apparently not effective in this case. Anthelmintics used and treatment schedule should be revised and small herds raised at households should also be included and treated.

Molecular diagnosis of natural fasciolosis by DNA detection in sheep faeces.

Fasciolosis is an important parasitic zoonosis considered the most important helminth infection of ruminants in tropical countries. The aim of this study was to develop a PCR assay for the sensitive and specific detection of F. hepatica in formalin preserved sheep faeces. A 405-bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica was amplified from stool samples of infected sheep. The PCR assay showed a detection limit of 20 pg of F. hepatica DNA. No cross-reactions were observed with samples containing coccidian oocysts or gastrointestinal nematodes eggs. Our PCR technique showed to be effective for specific detection of F. hepatica infections in sheep.