Origin of the natural variation in the storage of dietary carotenoids in freshwater amphipod crustaceans.

Carotenoids are diverse lipophilic natural pigments which are stored in variable amounts by animals. Given the multiple biological functions of carotenoids, such variation may have strong implications in evolutionary biology. Crustaceans such as Gammarus amphipods store large amounts of these pigments and inter-population variation occurs. While differences in parasite selective pressure have been proposed to explain this variation, the contribution of other factors such as genetic differences in the gammarid ability to assimilate and/or store pigments, and the environmental availability of carotenoids cannot be dismissed. This study investigates the relative contributions of the gammarid genotype and of the environmental availability of carotenoids in the natural variability in carotenoid storage. It further explores the link of this natural variability in carotenoid storage with major crustacean immune parameters. We addressed these aspects using the cryptic diversity in the amphipod crustacean Gammarus fossarum and a diet supplementation protocol in the laboratory. Our results suggest that natural variation in G. fossarum storage of dietary carotenoids results from both the availability of the pigments in the environment and the genetically-based ability of the gammarids to assimilate and/or store them, which is associated to levels of stimulation of cellular immune defences. While our results may support the hypothesis that carotenoids storage in this crustacean may evolve in response to parasitic pressure, a better understanding of the specific roles of this large pigment storage in the crustacean physiology is needed.

Renal Microsporidiosis in Pediatric Bone Marrow Transplant Recipients: A Case Series.

Microsporidiosis is a rare, but emerging opportunistic infection in solid organ transplant and stem cell transplant recipients. Renal involvement in microsporidiosis is very rarely seen in these recipients. We describe two cases of pediatric renal microsporidiosis, diagnosed on renal biopsies, following bone marrow transplantation presenting as severe acute kidney injury. The first patient died, whereas the second survived due to early diagnosis based on high index of suspicion and prompt treatment with Albendazole. We believe these are the first such reported cases of renal microsporidiosis in pediatric bone marrow transplant recipients.

Anncaliia algerae Microsporidial Myositis, New South Wales, Australia.

We describe the successful management of Anncaliia algerae microsporidial myositis in a man with graft versus host disease after hemopoietic stem cell transplantation. We also summarize clinical presentation and management approaches and discuss the importance of research into the acquisition of this infection and strategies for prevention.

Evaluation of two DNA extraction methods for the PCR-based detection of eukaryotic enteric pathogens in fecal samples.

OBJECTIVE: Efficient and easy-to-use DNA extraction and purification methods are critical in implementing PCR-based diagnosis of pathogens. In order to optimize the routine clinical laboratory diagnosis of eukaryotic enteric pathogens, we compare, via quantitative PCR cycle threshold (Ct) values, the efficiency of two DNA extraction kits: the semi-automated EZ1® (Qiagen) and the manual QIAamp® DNA Stool Mini Kit (Qiagen), on six protozoa: Blastocystis spp., Cryptosporidium parvum/hominis, Cyclospora cayetanensis, Dientamoeba fragilis, Giardia intestinalis and Cystoisospora belli and one microsporidia: Enterocytozoon bieneusi. RESULTS: Whereas EZ1® (Qiagen) and QIAamp® DNA Stool Mini Kit (Qiagen) yielded similar performances for the detection of Cryptosporidium spp. and D. fragilis, significant lower Ct values (p < 0.002) pointed out a better performance of EZ1® on the five remaining pathogens. DNA extraction using the semi-automated EZ1® procedure was faster and as efficient as the manual procedure in the seven eukaryotic enteric pathogens tested. This procedure is suitable for DNA extraction from stools in both clinical laboratory diagnosis and epidemiological study settings.

Microsporidia Intracellular Development Relies on Myc Interaction Network Transcription Factors in the Host.

Microsporidia are ubiquitous parasites that infect a wide range of animal hosts, and these fungal-related microbes undergo their entire replicative lifecycle inside of host cells. Despite being widespread in the environment and causing medical and agricultural harm, virtually nothing is known about the host factors important to facilitate their growth and development inside of host cells. Here, we perform a genetic screen to identify host transcription factors important for development of the microsporidian pathogen Nematocida parisii inside intestinal cells of its natural host, the nematode Caenorhabditis elegans Through this screen, we identified the C. elegans Myc family of transcription factors as key host regulators of microsporidia growth and development. The Mad-like transcription factor MDL-1, and the Max-like transcription factors MXL-1 and MXL-2 promote pathogen levels, while the Myc-Mondo-like transcription factor MML-1 inhibits pathogen levels. We used epistasis analysis to show that MDL-1 and MXL-1, which are thought to function as a heterodimer, appear to be acting canonically. In contrast, MXL-2 and MML-1, which are also thought to function as a heterodimer, appear to be acting in separate pathways (noncanonically) in the context of pathogen infection. We also found that both MDL-1::GFP and MML-1::GFP are expressed in intestinal cells during infection. These findings provide novel insight into the host transcription factors that regulate microsporidia development.

Intestinal microsporidiosis.

Infection by the ingested pathogens of microsporidia occur primarily in immunosuppressed patients (including untreated HIV/AIDS) and are diagnosed by stool examination, small bowel biopsy with special stains, or electron microscopy (for definitive speciation), or by various molecular techniques. Although electron microscopy has been the definitive diagnostic tool for speciation, genetic sequencing increasingly provides the definitive diagnosis for new species, such as Anncaliia algerae. Further genetic sequencing of the common pathogens may allow for the development of advanced molecular diagnostics providing high diagnostic sensitivity and throughput.

Microsporidiasis.

Microsporidia are obligate intracellular spore-forming organisms. Several species of microsporidia cause human disease, mainly in immunocompromised hosts. The spectrum of disease varies from diarrhea, keratoconjunctivitis to disseminated infection involving multiple organs. CNS disease is a rare manifestation usually seen in compromised hosts as part of a disseminated infection. Only 12 cases of CNS microsporidiosis have been reported in the literature. Clinically, they usually present with signs and symptoms of encephalitis and seizures. Diagnosis often requires brain biopsy, but spores can occasionally be found in other sites. Albendazole and fumagillin have been successfully used in treating microsporidiosis at other sites, but their role in CNS infection is unclear.

Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites.

Disseminated microsporidiosis in an immunosuppressed patient.

We report a case of disseminated microsporidiosis in a patient with multiple myeloma who had received an allogeneic stem cell transplant requiring substantial immunosuppression. The causative organism was identified as Tubulinosema acridophagus, confirming this genus of microsporidia as a novel human pathogen.

Infections of Blastocystis hominis and microsporidia in cancer patients: are they opportunistic?

Chemotherapy can cause immunosuppression, which may trigger latent intestinal parasitic infections in stools to emerge. This study investigated whether intestinal parasites can emerge as opportunistic infections in breast and colorectal cancer patients (n=46 and n=15, respectively) undergoing chemotherapy treatment. Breast cancer patients were receiving a 5-fluorouracil/epirubicin/cyclophosphamide (FEC) regimen (6 chemotherapy cycles), and colorectal cancer patients were receiving either an oxaliplatin/5-fluorouracil/folinic acid (FOLFOX) regimen (12 cycles) or a 5-fluorouracil/folinic acid (Mayo) regimen (6 cycles). Patients had Blastocystis hominis and microsporidia infections that were only present during the intermediate chemotherapy cycles. Thus, cancer patients undergoing chemotherapy should be screened repeatedly for intestinal parasites, namely B. hominis and microsporidia, as they may reduce the efficacy of chemotherapy treatments.

Host specificity of microsporidia pathogenic to the gypsy moth, Lymantria dispar (L.): field studies in Slovakia.

Several species of microsporidia are important chronic pathogens of Lymantria dispar in Europe but have never been recovered from North American gypsy moth populations. The major issue for their introduction into North American L. dispar populations is concern about their safety to native non-target insects. In this study, we evaluated the susceptibility of sympatric non-target Lepidoptera to two species of microsporidia, Nosema lymantriae and Vairimorpha disparis, isolated from European populations of L. dispar and applied in field plots in Slovakia. Application of ultra low volume sprays of the microsporidia maximized coverage of infective spores in a complex natural environment and, thus, exposure of non-target species to the pathogens. Of 653 non-target larvae collected from plots treated with V. disparis in 2002, 18 individual larvae representing nine species in four families were infected. These plots were monitored for two subsequent seasons and V. disparis was not recovered from non-target species. Of 2571 non-target larvae collected in N. lymantriae-treated sites, one larva was found to be infected. Both species of microsporidia, particularly N. lymantriae, appear to have a very narrow host range in the field, even when an inundative technique is used for their introduction. V. disparis infections in L. dispar exceeded 40% of recovered larvae in the treated study sites; infection rates were lower in sites sprayed with N. lymantriae. Several naturally-occurring pathogens were recorded from the non-target species. The most common pathogen, isolated from 21 species in eight families, was a microsporidium in the genus Cystosporogenes.

Effects of host temperature and gastric and duodenal environments on microsporidia spore germination and infectivity of intestinal epithelial cells.

Approximately 14 of the more than 1,000 species of microsporidia infect humans, only two of which, Enterocytozoon bieneusi and Encephalitozoon intestinalis, cause intestinal microsporidiosis. Clinical isolates of three microsporidia species, E. intestinalis, Encephalitozoon hellem, and the insect parasite, Anncaliia (Brachiola, Nosema) algerae were used in a spore germination assay, and enterocyte attachment and infection assays were performed to model the potential roles of gastric and duodenal environments and host temperature in determining why only one of these microsporidia species causes intestinal microsporidiosis. Enterocyte infection with A. algerae spores was 10% that of the Encephalitozoon species, a difference not attributable to differences in spore attachment to host cells. Prior spore treatment with pepsin in HCl, pancreatic enzymes, or ox bile did not inhibit germination or enterocyte infection by the three microsporidia species. While the Encephalitozoon species differentiated to mature spores within 3 days, the time taken for many enterocytes to turn over, A. algerae took 3-5 days to produce mature spores, near the upper limit for enterocyte turnover in vivo. Thus, host temperature may contribute to A. algerae not causing human intestinal microsporidiosis, but none of the factors tested account for the inability of E. hellem to cause such an infection.

Enterospora canceri n. gen., n. sp., intranuclear within the hepatopancreatocytes of the European edible crab Cancer pagurus.

Only 1 genus (Nucleospora) within 1 family (Enterocytozoonidae) of the Microsporidia contains species that are parasitic within the nuclei of their host cells; to date, all described intranuclear Nucleospora spp. parasitise fish. This study describes the first intranuclear microsporidian parasite of an invertebrate, the European edible crab Cancer pagurus L. (Decapoda: Cancridae). Infected crabs displayed no obvious external signs, and maximum apparent prevalence of infection within a monthly sample was 3.45%. Infected hepatopancreatic tubules were characterised by varying numbers of hypertrophic and eosinophilic nuclei within epithelial cells. Parasite stages appeared as eosinophilic granular accumulations causing margination of host chromatin. In advanced cases, the tubule epithelia degenerated, with parasites and sloughed epithelial cells appearing in tubule lumens. All life stages of the parasite were observed within host nuclei. Uninucleate meronts were not detected, although binucleate stages were observed. Multinucleate plasmodia (sporogonal plasmodia) contained up to 22 nuclei in section, and late-stage plasmodia contained multiple copies of apparatus resembling the polar filament and anchoring disk, apparently associated with individual plasmodial nuclei. As such, aggregation and early assembly of sporoblast components took place within the intact sporogonial plasmodium, a feature unique to the Enterocytozoonidae. Liberation of sporoblasts from plasmodia or the presence of liberated sporoblasts was not observed in this study. However, large numbers of maturing and mature spores (measuring 1.3 +/- 0.02 x 0.7 +/- 0.01 microm) were frequently observed in direct contact with the host nucleoplasm. Considering the shared features of this parasite with microsporidians of the family Enterocytozoonidae, and the unique presence of this parasite within the nucleoplasm of decapod crustacean hepatopancreatocytes, this parasite (Enterospora canceri) is proposed as the type species of a new genus (Enterospora) of microsporidian. Molecular taxonomic work is now required, comparing Enterospora to Enterocytozoon and Nucleospora, the 2 other genera within the Enterocytozoonidae.

Enterospora sp., an intranuclear microsporidian infection of hermit crab Eupagurus bernhardus.

Recent work at our laboratory has led to the discovery of a new genus of microsporidian parasite residing in the family Enterocytozoonidae. The type species of this new genus, Enterospora canceri, is an intranuclear parasite infecting the hepatopancreatocytes of the decapod crustacean Cancer pagurus. Here we provide the second description of a parasite within the genus Enterospora, this time infecting the hermit crab Eupagurus bernhardus from U.K. waters. The pathological manifestation and ultrastructural features of the hermit crab parasite are very similar to those described for E. canceri. Further taxonomic comparisons based upon ultrastructural and molecular affinities of Enterospora are now required to define firmer links between this new genus within the Enterocytozoonidae and all other microsporidian families. The opportunistic nature of the discovery of a second intranuclear microsporidian within the Crustacea suggests that their presence may be more common than in higher animal groups.

Esporos de microsporidios en heces de dos niños HIV seronegativos de Tucumán, Argentina / Microsporidian spores in the stool specimens of two HIV-negative children from Tucuman, Argentina

La incidencia mundial de microsporidiosis intestinal en pacientes infectados por HIV es relevante. En contraste, son muy pocos los casos de infección del parásito en pacientes HIV negativos. Aquí se informa que en diciembre de 1994 fueron detectadas infecciones microsporidianas intestinales en dos niños, hermanos, de 16 y 33 meses de edad, HIV-negativos, desnutridos, nativos y residentes en la provincia de Tucumán, Argentina. El diagnóstico se logró empleando microscopía óptica para la detección de esporos microsporidianos en frotis de especímenes fecales coloreados con una nueva modificación del método de Ziehl-Neelsen modificado. El reconocimiento de los esporos estuvo basado en sus características morfológicas y propiedades tintoriales, y en su comparación con frotis testigo positivo para Enterocytozoon bieneusi coloreados con el método de Weber. Ambos niños mostraron definidos signos de desnutrición y otras patologías asociadas, lo que sumado a la falta de colaboración de sus progenitores para un adecuado estudio clínico, hicieron dificultoso poner en claro el rol desempeñado por los parásitos en estos casos. No obstante, las infecciones microsporidianas de los dos niños tuvieron evolución autolimitada

Microsporidiosis humana / Human microsporidiosis

La microsporidiosis humana ha cobrado relevancia clínica con la aparición del virus de la inmunodeficiencia humana. La diarrea crónica constituye la expresión clínica más frecuente es estos pacientes aunque se han descrito infecciones localizadas en los distintos parénquimas del organismo e infecciones generalizadas. Las especies más relevantes son enterocytozoon bieneusi y encephalitozoon intestinalis. Se plantean como desafíos definir claramente los métodos diagnósticos y terapia adecuadapara ambas especies, una eventual terapia profiláctica primaria y/o secundaria y actualizar además las cifras de prevalencia en los distintos países

Enterocytozoon bieneusi (orden microsporidia, familia entrerocytozoonidae) in Costa Rica: report of the first human case in central america

The first case of microspotidiosis in Central America is describes in en AIDS patient from Costa Rica. Electronic microscopy studies indicate that the spores were not included in a parasitophorous vacuole, but they are in direct contact with the cell cytoptasm. Sporogonic proliferative plasmodial forms presence and localization of the polar tubes in the anterior region of the spore, confirmed the specie Enterocytozoon bieneusi as the cause of this microsporidian infection

Microsporidioses humanas: desenvolvimento de métodos para o diagnóstico morfológico das microsporidioses intestinais em pacientes HIV-Positivos no município do Rio de Janeiro / Microsporídiosis human: development of methods for the morphological diagnosis of intestinal microsporídiosis in HIV-positives in the municipality of Rio de Janeiro

As infecções intestinais por Enterocytozoon bieneuse e Septata intestinalis têm sido frequêntemente diagnosticadas em pacientes com SIDA/AIDS. Entretanto a detecção de esporos de microsporídios nas fezes é particularmente difícil quando o número dessas estruturas de resistência é pequeno. A partir de um método específico para a detecção de oocistos de Cryptosporidium, desenvolveu-se neste trabalho uma modificação que permitiu a concentração de esporos de microsporídios nas fezes. Foram estudados 140 pacientes do Hospital Evandro Chagas/FIOCRUZ e 95 do Hospital Universitário Pedro Ernesto/UERJ, todos soropositivos para o vírus da imunodeficiência humana(HIV). As amostras fecais desses pacientes foram investigadas pelo método direto e pelo método de concentração, desenvolvido neste trabalho. Todas as lâminas foram coradas pelas técnicas do Chromotrope e do Gram-Chromotrope, sendo essa última coloração desenvolvida e testada durante os trabalhos experimentais. Foram obtidas prevalências++. para microsporídios de 17,86 por cento (método direto) e 25 por cento (método de concentração) no grupo de indivíduos do HEC/FIOCRUZ e de 11,58 por cento no grupo investigado do HUPE/UERJ. A microscopia eletrônica de transmissão, realizada em fragmentos de biópsia duodenal de um paciente com diagnóstico positivo pela microscopia óptica, permitiu a identificação de esporos que apresentaram ultra-estrutura caracter161stica, compatível com a espécie E. bieneusi. O número de pacientes que foi diagnosticado com microsporidiose intestinal no presente estudo mostrou que os microsporídios são importantes agentes de infecção em indivíduos portadores do vírus HIV no Município do Rio de Janeiro

Microsporidioses humanas: desenvolvimento de métodos para o diagnóstico morfológico das microsporidioses intestinais em pacientes HIV-Positivos no município do Rio de Janeiro

As infecções intestinais por Enterocytozoon bieneuse e Septata intestinalis têm sido frequêntemente diagnosticadas em pacientes com SIDA/AIDS. Entretanto a detecção de esporos de microsporídios nas fezes é particularmente difícil quando o número dessas estruturas de resistência é pequeno. A partir de um método específico para a detecção de oocistos de Cryptosporidium, desenvolveu-se neste trabalho uma modificação que permitiu a concentração de esporos de microsporídios nas fezes. Foram estudados 140 pacientes do Hospital Evandro Chagas/FIOCRUZ e 95 do Hospital Universitário Pedro Ernesto/UERJ, todos soropositivos para o vírus da imunodeficiência humana(HIV). As amostras fecais desses pacientes foram investigadas pelo método direto e pelo método de concentração, desenvolvido neste trabalho. Todas as lâminas foram coradas pelas técnicas do Chromotrope e do Gram-Chromotrope, sendo essa última coloração desenvolvida e testada durante os trabalhos experimentais. Foram obtidas prevalências++. para microsporídios de 17,86 por cento (método direto) e 25 por cento (método de concentração) no grupo de indivíduos do HEC/FIOCRUZ e de 11,58 por cento no grupo investigado do HUPE/UERJ. A microscopia eletrônica de transmissão, realizada em fragmentos de biópsia duodenal de um paciente com diagnóstico positivo pela microscopia óptica, permitiu a identificação de esporos que apresentaram ultra-estrutura caracter161stica, compatível com a espécie E. bieneusi. O número de pacientes que foi diagnosticado com microsporidiose intestinal no presente estudo mostrou que os microsporídios são importantes agentes de infecção em indivíduos portadores do vírus HIV no Município do Rio de Janeiro

Actualizaciones sobre microsporidiosis en el hombre / Update on microsporidiosis in man

The importance of microsporidium as an opportunistic agent in immunocompromised and AIDS patients is reviewed. Five strains of the agent have been described: Encephalitozoon, Enterocytozoon, Nosema, Pleistophora and Septata. The clinical presentation may be as 1) Generalized infections with multisystemic involvement, specially of the central nervous system; 2) Intestinal, that is the most important and frequent localization in man, and that may cause death in AIDS patients; 3) Ocular, that affects cornea, conjunctiva and may extend to paranasal sinuses; 4) Liver and biliary tract infection with granulomatous lesions, hepatic necrosis or sclerosing colangitis and 5) Muscular, affecting skeletal muscle. The diagnosis is difficult and is established finding spores in the affected tissues with light or electron microscopy. Lately, the diagnosis of intestinal microsporidiosis is made looking for faecal spores. The resistant wall of spores hampers treatment. However, good results are obtained with albendazole in intestinal microsporidiosis