microsporidia.pptx

About microsporum
Life cyle
Importance
Diseases

1. Parasite Authors: Dr C . del Águila de la Puente, Dr S . Fenoy , Dr A . Magnet , M. Dr D . O. Baceiredo , Dr F . Izquierdo.

2. 1. Infectious Agent and Morphology

3. Microsporidia are obligate intracellular parasites with a unique mode of entering host cells via a polar tube. They are true eukaryotes with a nucleus, chromosome separation and an intracytoplasmic membrane system. However, they lack a classical stacked Golgi apparatus, centrioles, peroxisomes and the mitochondria have been substituted by a mitochondria-like organelle called mitosome ( Franzen and Muller 1999; Keeling 2011). Microsporida have a unique structure in nature, the polar tube or polar filament that is involved in host cell invasion ( Bigliardi and Sacchi 2001). Their infective stage is the spore ( Figure 1.1 ), ranging 1-20 µm long, but species infecting mammals are smaller (1-3µm), Encephalitozoon sp spores measure 2.0 to 2.5 by 1.0 to 1.5 µm, and Enterocytozoon bieneusi spores measure 0.7 to 1 by 1-1,6  m . Figure 1.1 : Morphology and diagram of a microsporidian spore. (Parasitology Laboratory, Universidad San Pablo-CEU). 1.1 Level Classification and 1.2 Morphology 1 Microsporidia Scientific classification SuperKingdom Eukaryota Kingdom Protozoa Phylum Microsporidia

4. 1.2 Morphology 2 They are gram-positive and environmentally resistant, with a thick wall composed by three layers: an electron-dense proteinaceus outer layer, called exospore , an electron-lucent inner chitinous layer called endospore , and a plasma membrane enclosing an infective sporoplasm ( Franzen and Muller 1999, Vavra 1999). The content of the spores is composed of two functionally different parts: the sporoplasm and the extrusion apparatus . The sporoplasm may be considered as the infectious material of microsporida . It may contain a single nucleus , as it is the case of Encephalitozoon and Enterocytozoon , or two nucleus in Nosema , Brachiola and Vittaforma , which consist of two closely apposed nuclei functioning as a single unit ( Bigliardi and Sacchi 2001), with ribosomes and endoplasmic reticulum membranes ( Delbac and Polonais 2008).

5. 1.2 Morphology 3 The extrusion apparatus is composed of a polar tube, coiled around the posterior region of the spore, an anchoring disk, an anterior membrane-bounded organelle, termed polaroplast , and a vacuole at the posterior end ( Figure 1.2 ). The number and disposition of polar filament coils vary among microsporidia, showing 5 to 7 coils in a single row in Encephalitozoon species and Enterocytozoon with double row. Under certain conditions such as alkaline pH, or increased concentrations of Na + , K + , Cl - , Ca 2+ ions, the spore germinates and an inflow of water dramatically increases the pressure inside the spore. Figure 1.2: Life cycle of microsporidia. Infection phase (A ), Sporogony phase ( B) . (Parasitology Laboratory, USP-CEU).

6. 2. Life Cycle

7. 2.1 Life Cycle Overview This eventually ruptures the wall at the anterior end, where the exospore is less thick, forcing the polar filament to eject turning inside out to form a tube ( Delbac and Polonais 2008; Keeling 2009). Germination is a very quick process: the polar tube acts as a projectile if penetrates the host cell membrane, it delivers the sporoplasm inside the host cell cytoplasm ( Figure 2.1A ). Once inside the host cell cytoplasm, microsporidian proliferation ( merogony ) will occur within a parasitophorous vacuole (as in the case of Encephalitozoon species), or in direct contact with the host cell cytoplasm (e.g. Enterocytozoon ). At the end of the proliferative phase ( Figure 2.1B ), the cytoplasm of the host cell is completely filled with spores and the cell membrane will disrupt releasing the mature spores that can infect new cells to continue the life cycle ( Franzen and Muller 1999; Bigliardi and Sacchi 2001; Delbac and Polonais 2008). Figure 2.1: Life cycle of microsporidia. Infection phase (A), Sporogony phase (B) . (Parasitology Laboratory, USP-CEU).

8. 3. Host Parasite Relationship and Immune Response

9. 3.1 Epidemiology 1 To date, the knowledge of the real human Encephalizoon and Enterocytozoon infections is not easy to discern, as they are often misdiagnosed or overlooked. E. intestinalis has been described with variable presence in all continents depending on the country. However, in general, in HIV+ patients, varies from 0.9% in Switzerland (Coyle et al. 1996) to 12.8% recently detected in Russian HIV+ patients. In this case, the prevalence was unexpectedly higher than that observed for E. bieneusi ( Sokolova et al. 2011). However, the data on immunocompetent individuals are still scarce. It has been associated mainly to travellers' diarrhoea although serologic studies have shown high seroprevalence up to 8% (Raynaud et al. 1998; Wichro et al. 2005; Mathis, Weber, and Deplazes 2005). The zoonotic potential of these species has been recognized, since it has been occasionally described in a great variety of mammals, such as donkeys, dogs, pigs, cows, goats and recently in cats and avian hosts so, the most likely source of this infection may be other infected humans and animals (Didier 2005; Mathis, Weber, and Deplazes 2005; Velasquez et al. 2012). Figure 3.1: Encephalitozoon intestinalis spores from cell culture. Spores detected by IFAT with polyclonal antibodies. The arrows mark the extruded polar tubes. (Parasitology Laboratory, USP-CEU).

10. 3.1 Epidemiology 2 Water-borne transmission must be considered, since the spores are environmentally resistant and can survive in water. For example, in spite of their small size, recent publications have detected the presence of E. intestinalis in water (Dowd, Gerba , and Pepper 1998; Izquierdo et al. 2011; Galvan et al. 2013). These spores have been concentrated using the IDEXX Filta -Max ® , recommended by the USEPA, to detect Cryptosporidium and Giardia in water (U.S.E.P.A 2005; Izquierdo et al. 2011). In the case of E. intestinalis infections, an oral-faecal transmission route is indicated, given the multiple sites of infection and that spores can be released via stools. Other modes of infection could be inhalation and contaminated fingers, although waterborne route must be taken in consideration, since spores of E. intestinalis have been detected in recreational water (Dowd, Gerba , and Pepper 1998; Didier and Weiss 2006; Izquierdo et al. 2011). Human infections by E. cuniculi are traditionally described as less frequent than E. intestinalis infections in studies carried out with HIV+ patients and in patients with severe immunosuppression, in Europe and the USA, revised in Talabani et al. (2010) and recently, in Africa ( Ojuromi et al. 2012). However, studies on immunocompetent individuals have shown a prevalence higher than expected indicating that human exposure may be more common than previously suspected. Figure 3.2: Natural bath (Madrid, Spain) (Parasitology Laboratory, USP-CEU).

11. 3.1 Epidemiology 3 In the case of E. hellem infections, reports in humans are rare. Around 50 HIV + patients infected with E. hellem have been described in Europe, United States and Africa (Mathis, Weber, and Deplazes 2005). However, recent studies in immuncompetent individuals have shown higher prevalence ( Sak , Brady et al. 2011). This species has been described mainly in avian hosts, such as parrots, ostriches, peach-face lovebirds and pigeons, some of them closed to humans and is considered of zoonotic origin (Black et al. 1997; Gray, Puette , and Latimer 1998; Snowden, Logan, and Phalen 2000; Haro et al. 2005). The parrots or pigeons excrete spores in faeces which, although not yet demonstrated, suggest that birds may be a source for the zoonotic origin of this microsporidium. Finally in the case of E. bieneusi , the immunocompromised and immunocompetent populations are affected with this infection, with data on microsporidia presence not only in HIV/AIDS patients but also in HIV-negative patients including travellers, the elderly, organ transplant recipients, Crohn’s disease patients and the immunocompetent population. Similar to data described worldwide, E. bieneusi is the species most frequently identified. Animal hosts including dogs, goats and rabbits, pigeons and soil and faecal samples (presumably from cats and dogs) from urban parks have also been described for microsporidia in several regions of Spain, with E. bieneusi and Encephalitozoon hellem as the species identified. All these data suggest that human pathogenic microsporidia circulate in the environment in Spain and support the idea that the most frequent microsporidia associated with human infection are of zoonotic origin ( Galván-Díaz , 2014). Figure 3.3: Enterocytozoon bieneusi spore in stool sample. Spore stained with Modified Trichrome. The arrow mark the spore. (Parasitology Laboratory, USP-CEU).

12. 4. Clinical and Pathological Features

13. 4.1 Clinical Features Clinical features of microsporidiosis due to Encephalitozoon sp and Enterocytozoon are broad because to the possibility to disseminate and produce systemic diseases. Included among the variety of manifestations are intestinal, ocular, renal and pulmonary disorders, although the immune status of the host is important in the clinical course of the disease (Didier 2005). Clinically silent chronic infections are usually developed in immunologically competent host, even though clinical signs could be developed after early infections (Snowden et al. 1998; Didier 2005; Sak , Kvac et al. 2011). In immunocompromised hosts, microsporidial infections develop serious diseases that may produce death. The most common clinical symptoms are chronic diarrhoea and malabsorption although, systemic diseases can be developed. Due to their dissemination capacity these parasites have been detected in numerous varieties of locations producing a great variety of manifestations.

14. 4.2 Microsporidial Diarrhoea and Biliary Pathology E . intestinalis and Enterocytozoon are the species more frequently implicated in intestinal and biliary pathology. Chronic diarrhoea and extreme slimming are frequent in immunocompromised patients. The diarrhoea is not bloody, there are no fever, anorexia is joined to abdominal pain, sickness and vomiting. Intestinal malabsorption is frequent but unspecific. A case with a small bowel perforation has been described (Soule et al. 1997). E . cuniculi and E. hellem have only occasionally been found in the intestine of inmuno -compromised patients (del Aguila , Moura et al. 2001; Garcia 2007; Chabchoub et al. 2009). However, whether the presence of spores in faecal samples in these patients could be related with an actual infection, or on the contrary, be the result of spores passing through the intestine, inhaled or ingested, is under discussion ( Haro et al. 2005; Graczyk et al. 2007). It is important to note that spores are shed primarily in urine rather than faeces (Didier and Weiss 2011). 4.3 Microsporidial Hepatitis and Peritonitis The cases described are scarce and isolates and have been attributed to E. intestinalis and E. hellem . To date, only two cases have been reported with hepatic involvement, one case the hepatitis was described at autopsy produced by E. cuniculi (Terada et al. 1987) and in the second case, a fulminant hepatic failure was referenced in a HIV+ patient with previous diarrhoea lasting 2 months (Sheth et al. 1997). Peritonitis is an unusual presentation of microsporidia infections.

15. 4.4 Disseminated Infections The three Encephalitozoon species and Enterocytozoon bieneusi have been described in immunocompromised patients, and in transplant recipients with multiple–organ failure, although they can also produce disseminated infections in the immunocompetent (Weber et al. 1994; Cali, Kotler, and Orenstein 1993; Galvan et al. 2011; Ditrich et al. 2011; Galván-Díaz et al. 2011). The clinical manifestations are variable. Beyond gastrointestinal manifestations, keratoconjuntivitis , bronquiolitis , sinusitis, nephritis, urethritis, cystitis, prostatitis, hepatitis and peritonitis manifestations are frequent ( Figures 4.1a and 4.1b ). Other organs can be involved such as brain and genital tract (Weber et al. 1994; Mohindra et al. 2002; Didier 2005; Torres et al. 2012 ). E. intestinalis has been associated to enteric disease, although it can infect kidneys, gallbladder, eyes, nasal mucosa, and skin. This microsporidium has been detected in saliva, urine and bronchoalveolar lavage fluid. E. hellem has been described as cause of pulmonary disease, keratitis/ ketratoconjuntivitis , kidney disease and nasal polyps. Finally, E. cuniculi can infect intestine, liver, peritoneum, kidneys, brain and eyes (Weber et al. 1994; Kotler and Orenstein 1998; Anane and Attouchi 2010; Ditrich et al. 2011; Robinson et al. 2011). Figure 4.1a: Parasitophorous vacuoles of Microsporidia in tissues (40X). spleen , spores detected by immunological technique (IFAT). (Parasitology Laboratory, USP-CEU). Figure 4.1b: Parasitophorous vacuoles of Microsporidia in tissues (40X). lung , stained spores with Gram- Chromotrope . (Parasitology Laboratory, USP-CEU).

16. 4.5 Ocular Microsporidiosis Ocular infections due to microsporidia have been described in both immunocompetent and immunocompromised patients and have recently received considerable attention ( Loh et al. 2009). This infection is mainly associated to E. hellem where a superficial punctate keratoconjuntivitis was described (Didier et al. 1991). Although, these manifestations have been described in inmunocompromised individuals or in contact lens wearers, recent studies indicated that this manifestation can occur in immunocompetent individuals (Chan et al. 2003; Sridhar and Sharma 2003). 4.6 Pathology and Immunology 1 Studies on the pathology of Encephalitozoon and Enterocytozoon have been carried out in immunocompromised patients, especially in AIDS patients. In the case of E. intestinalis infection, although the parasite infects primarily small-intestinal enterocytes, it may affect the perinuclear zone of intestinal lamina propria of macrophages, fibroblasts and endothelial cells (Weber et al. 1994; Conteas et al. 2000). It seems that via infected macrophages, the parasite can disseminate, to liver, pancreas, colon, lungs sinuses, kidneys, and conjunctiva (Orenstein, Dieterich , and Kotler 1992; Weber et al. 1994). In the intestine, a severe ulceration of the small bowel with mucosal atrophy, along with acute and chronic inflammation, has been described. Bile ducts infections are related to papillary stenosis, bile duct dilatation, alithiasic cholecystitis, and sclerosing cholangitis (Weber et al. 1994). Alterations produced by dissemination of the E. intestinalis , E. hellem and E. cuniculi out of intestine are related with the tissue affected. An inflammatory reaction has been observed in the liver, pancreas, lung, kidneys and eyes. In the case of respiratory tissues infection parasites have been detected in lining epithelium of almost the entire length of the tracheobronchial tree (Schwartz et al. 1992).

17. 4.6 Pathology and Immunology 2 The immunity against these parasites depends mainly on cell-mediate immunity. This fact was suspected from the beginning, when clinicians observed that patients with severe AIDS and with low CD4 + T cells showed a more severe manifestation of infection. Recent studies in animal models have shown that protective immunity is mainly dependent on CD8 + T cells. In contrast, the role of CD4 + cells does not seem necessary to control the infection, and the lack of CD4 + T cells does not affect induction CD8 + T cells ( Moretto et al. 2000; Ghosh and Weiss 2009). In the case of humoral immunity it is known that the infection activates antibody production and their presence is related with a latent infection. However, their presence alone does not look to have a protective role, although a study carried out in mice experimentally infected with E. cuniculi has shown that antibodies can contribute to resistance (Weber et al. 1994; Sak et al. 2006).

18. 5. Diagnosis

19. 5.1 Diagnosis and Detection The diagnosis in the case of human infection and the detection in environmental samples, is based on the observation of the characteristics spores. However, due to the small size of spores, multiple and specific diagnostic methods are required. Originally microsporidia identification was based on electron microscopic examination to determine the genus species, although not always reaching the species level. Nowadays, that method has been substituted by new staining methods for light microscopy (more available in diagnostic laboratories), immunofluorescence procedures and molecular test. ELISA IFAT TEM Figure 5.1: Pictures from Parasitology Laboratory, USP-CEU. M DNA Sample C+ C- PCR Cell Culture

20. 5.2 Electron Microscopy Electron microscopy was considered the gold standard to confirm the diagnosis of microsporidiosis and specie identification, based in the characteristic of the polar tube ( Figures 5.2a and 5.2b ). However compared to others, this method is less sensitive and is not available in most laboratories for clinical diagnosis. Nowadays, it is limited to taxonomic studies. Figure 5.2a : Immunogold electron micrographs of Encephalitozoon intestinalis spores. (Arrows mark the polar tube .) (Parasitology Laboratory, USP-CEU). Figure 5.2b : Immunogold electron micrographs of Encephalitozoon intestinalis spores. (Arrows mark the polar tube .) Parasitology Laboratory, USP-CEU).

21. 5.3 Staining Methods Staining methods for microsporidia detection include Modified Trichrome stains (Weber et al. 1992). The spore wall should stain pinkish to red, with the interior of the spore being clear or showing a horizontal or diagonal stripe, which represents the polar tube ( Figure 5.3a ). This method can be used mainly for stools and body fluids. In the case of tissue sections a Gram- chromotrope staining is recommended ( Visvesvara and Garcia 2002) and could be used in stools samples too ( Figure 5.3b ). It does not allow the species identification. Figure 5.3a : Microsporidia spores stained in faecal smear . Enterocytozoon bieneusi (Modified Trichrome). ( Gram- Chromotrope ). (Parasitology Laboratory, USP-CEU). Figure 5.3b : Microsporidia spores stained in faecal smear . Encephalitozoon intestinalis (Gram- Chromotrope ). (Parasitology Laboratory, USP-CEU).

22. 5.4 Fluorescent Staining Techniques Fluorescent staining techniques using optical fluorochrome is another staining method used, in addition to Modified Trichrome stain to confirm microsporidia spores. This method is based in the capacity of some chemo-fluorescent agents to stain chitin, the main component of the wall spores ( Calcofluor White, or Uvitex 2B) (Garcia 2002) and allows the detection of spores that could be missed by the Trichrome-stain ( Figure 5.4 ). Figure 5.4: Encephalitozoon intestinalis spores in fecal smear stained by the Calcofluor technique. (Parasitology Laboratory, USP-CEU).

23. 5.5 Immunofluorescent Test Species determination can be carried out with the use of indirect immunofluorescence test ( Figures 5.5A and 5.5B ). This method has been improved since the development of monoclonal antibodies against the different species of Encephalitozoon sp (Hoffman et al. 2007; Li et al. 2011; Peer et al. 2012). It has been described as having high sensibility and specificity and comparable to PCR methods, the most used routinely in spite of their high cost and complexity ( Polley et al. 2011). Figure 5.5: Encephalitozoon cuniculi spores from cell culture. A and B: Spores detected by IFAT with polyclonal antibodies. The arrows mark the extruded polar tubes. (Parasitology Laboratory, USP-CEU) A B

24. 5.6 PCR Methods The genetic diagnosis has been increasingly employed because it allows the detection of small numbers of spores or other stages of the parasite that may be undetected under the microscope. Molecular techniques are methods that provide the species identification, although routinely used in clinical diagnosis, to date there are no commercial kits available, and their use is limited to specialized laboratories. During the last year, PCR-based methods have been increasingly used in order to improve sensitivity and specificity ( Figure 5.6 ). The application of PCR in clinical and environmental samples is subject to limitations such as the viable concentration of spores and the presence of inhibitors of the PCR. The latter is of special interest in the case of stool samples and water proceeding from wastewater sources. Furthermore, the sensibility of this technique is much higher than optical microscopy (Menotti et al. 2003). B 100 pb -- 600 pb -- M C + C- DNA Sample Figure 5.6: PCR amplification of rDNA coding region of the ITS of Encephalitozoon intestinalis .

25. 5.7 Cell Culture All the species of Encephalitozoon can be cultured in vitro but cell culture is not a routine method in diagnostic laboratories. Nevertheless, in vitro culture can provide information: the culture mass can be used to immunological, biochemical, physiological and molecular characterization studies, and even for the development of immunological or molecular reagents as well as different studies on the efficacy of antimicrobial agents. A great variety of cell lines has been used proceeding from monkey ( Figure 5.7 ), rabbit and human (Garcia 2002). Unfortunately, one of the most common human microsporidial pathogens, E. bieneusi , has been cultured only in short-term cultures lasted anywhere from 6 weeks to 6 months ( Visvesvara , 2002). Figure 5.7: Encephalitozoon cuniculi cell culture (Vero-E6). The arrows mark several parasitophorous vacuoles. (Parasitology Laboratory, USP-CEU).

26. 6. Treatment and Prevention

27. 6.1 Treatment The treatment of microsporidiosis is an unsolved problem, as it is an intracellular parasite, and also, to the resistance of spores. Since the discovery of microsporidia as infectious agents, a number of drugs has been used with variable effectiveness. In the case of intestinal and disseminated (non-ocular) Encephalitozoon infections, Albendazole , a Benzimidazole that inhibits tubulin polymerization and has antihelminthic and antifungal activity, is the drug of choice, as demonstrated by the complete elimination of the parasite after 7 days of treatment (Didier 2005; Anane and Attouchi 2010; Robinson et al. 2011). However, Albendazole is less-than optimal therapy in HIV patients with E. bieneusi and not result in clearance of the organisms from the stools. Fumagillin , an antibiotic and antiangiogenic is more broadly effective against Encephalitozoon spp. and E. bieneusi , however, is toxic when administered systemically to mammals (Didier, 2005). Since the use of antiretroviral therapy (HAAT), available in developed countries, a reduction in the prevalence of opportunistic infections, including microsporidiosis , has been observed without use of a specific treatment, simply by induction of a progressive reconstitution of the immune system. However, we remember that the different situation in developing countries, where the rapid expansion of AIDS, together with limited access to HAART, has contributed to an increased incidence of this disease ( Ojuromi et al. 2012).

28. 6.2 Prevention Since the sources of human infection are uncertain, specific measures of prevention are difficult to establish. Primary infection can occur by inhalation or ingestion of spores from environmental sources or by zoonotic transmission, suggesting the need of adequate hygiene rules, especially in the case of high-risk patients (HIV-positive patients, transplant recipients, elderly and children) (Garcia 2007). The measures included all those developed to prevent any infection of fecal -oral transmission related to hands, water and food ingestion (Didier 2005; Garcia 2007). Moreover, since clinical encephalitozoonosis is related with the immune-status of the host, in the case of HIV+ patients, reconstitution of immune-system by the use of HAART is an important measure of prevention ( Anane and Attouchi 2010). Figure 6.1: Natural bath (Madrid, Spain) (Parasitology Laboratory, USP-CEU).

29. 7. Public Health Risks

30. 7.1 Overview Measures related to animal contact should be developed, since one of the sources of human infection is animal-to-human (Didier 2005). Of interest is the description of the presence of E. hellem in park pigeons in their relation with humans, mainly children and the elderly people, or other immunosuppressed people. ( Haro et al. 2005). The presence of microsporidia related to waterborne transmission, has been documented. Studies in different types of water, including ditch and raw water, water treatment effluents, surface and ground water, irrigation and river water, recreational lakes, and wastewater, have shown the presence of Encephalitozoon sp and E. bieneusi ( Graczyk et al. 2007; Galvan et al. 2013). Their viability after treatment of drinking water and wastewater has been demonstrated ( Graczyk et al. 2004). Wastewater requires special attention, since this type of water can be discharged to a river or can be used for urban, agricultural, industrial, recreative and environmental practices, and may contribute to the contamination of the environment. These observations suggest that source of infection and modes of transmission require more definitive information in order to take measures to avoid health risks from microsporidia.

31. References

32. A-Con Anane , S., and H. Attouchi . 2010. Microsporidiosis : epidemiology, clinical data and therapy. Gastroenterol Clin Biol 34 (8-9):450-64. Bigliardi , E., and L. Sacchi . 2001. Cell biology and invasion of the microsporidia. Microbes Infect 3 (5):373-9. Black, S. S., L. A. Steinohrt , D. C. Bertucci , L. B. Rogers, and E. S. Didier. 1997. Encephalitozoon hellem in budgerigars ( Melopsittacus undulatus ). Vet Pathol 34 (3):189-98. Cali, A., D. P. Kotler, and J. M. Orenstein. 1993. Septata intestinalis N. G., N. Sp., an intestinal microsporidian associated with chronic diarrhea and dissemination in AIDS patients. J Eukaryot Microbiol 40 (1):101-12. Chabchoub , N., R. Abdelmalek , F. Mellouli , et al. 2009. Genetic identification of intestinal microsporidia species in immunocompromised patients in Tunisia. Am J Trop Med Hyg 80 (1):24-7. Chan, C. M., J. T. Theng , L. Li, and D. T. Tan. 2003. Microsporidial keratoconjunctivitis in healthy individuals: a case series. Ophthalmology 110 (7):1420-5. Conteas , C. N., O. G. Berlin, L. R. Ash, and J. S. Pruthi . 2000. Therapy for human gastrointestinal microsporidiosis . Am J Trop Med Hyg 63 (3-4):121-7.

33. Coy-Did Coyle , C. M., M. Wittner , D. P. Kotler, et al. 1996. Prevalence of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon ( Septata ) intestinalis among patients with AIDS-related diarrhea: determination by polymerase chain reaction to the microsporidian small-subunit rRNA gene. Clin Infect Dis 23 (5):1002-6. del Aguila , C., H. Moura , S. Fenoy , et al. 2001. In vitro culture, ultrastructure, antigenic, and molecular characterization of Encephalitozoon cuniculi isolated from urine and sputum samples from a Spanish patient with AIDS. J Clin Microbiol 39 (3):1105-8. Delbac , F., and V. Polonais . 2008. The microsporidian polar tube and its role in invasion. Subcell Biochem 47:208-20. Didier, E. S. 2005. Microsporidiosis : an emerging and opportunistic infection in humans and animals. Acta Trop 94 (1):61-76. Didier, E. S., and L. M. Weiss. 2006. Microsporidiosis : current status. Curr Opin Infect Dis 19 (5):485-92. Didier, E. S., and L. M. Weiss. 2011. Microsporidiosis : not just in AIDS patients. Curr Opin Infect Dis 24 (5):490-5.

34. Did-Gar Didier , E. S., P. J. Didier, D. N. Friedberg, et al. 1991. Isolation and characterization of a new human microsporidian, Encephalitozoon hellem (n. sp.), from three AIDS patients with keratoconjunctivitis . J Infect Dis 163 (3):617-21. Ditrich , O., A. Chrdle , B. Sak , et al. 2011. Encephalitozoon cuniculi genotype I as a causative agent of brain abscess in an immunocompetent patient. J Clin Microbiol 49 (7):2769-71. Dowd, S. E., C. P. Gerba , and I. L. Pepper. 1998. Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi , Encephalitozoon intestinalis, and Vittaforma corneae in water. Appl Environ Microbiol 64 (9):3332-5. Franzen , C., and A. Muller. 1999. Molecular techniques for detection, species differentiation, and phylogenetic analysis of microsporidia. Clin Microbiol Rev 12 (2):243-85. Galvan, A. L., A. M. Sanchez, M. A. Valentin, et al. 2011. First cases of microsporidiosis in transplant recipients in Spain and review of the literature. J Clin Microbiol 49 (4):1301-6. Galvan , A. L., A. Magnet , F. Izquierdo, et al. 2013. Molecular Characterization of Human-Pathogenic Microsporidia and Cyclospora cayetanensis Isolated from Various Water Sources in Spain: a Year-Long Longitudinal Study. Appl Environ Microbiol 79 (2):449-59. Garcia, L. S. 2002. Laboratory identification of the microsporidia. J Clin Microbiol 40 (6):1892-901 .

35. Gar-Hof Garcia , L. S.. 2007. Intestinal Protozoa ( Coccidia and Microsporidia) and Algae. In Diagnostic Medical Parasitology, Fifth Edition, ed. Lynne Shore García , 355-403. Washington D. C: ASM Press :57-101. Ghosh, K., and L. M. Weiss. 2009. Molecular diagnostic tests for microsporidia. Interdiscip Perspect Infect Dis 2009:926521. Graczyk , T. K., D. B. Conn, F. Lucy, et al. 2004. Human waterborne parasites in zebra mussels ( Dreissena polymorpha ) from the Shannon River drainage area, Ireland. Parasitol Res 93 (5):385-91. Graczyk , T. K., M. A. Johansson, L. Tamang , et al. 2007. Retrospective species identification of microsporidian spores in diarrheic fecal samples from human immunodeficiency virus/AIDS patients by multiplexed fluorescence in situ hybridization. J Clin Microbiol 45 (4):1255-60. Gray, M. L., M. Puette , and K. S. Latimer. 1998. Microsporidiosis in a young ostrich ( Struthio camelus ). Avian Dis 42 (4):832-6. Haro, M., F. Izquierdo, N. Henriques -Gil, et al. 2005. First detection and genotyping of human-associated microsporidia in pigeons from urban parks. Appl Environ Microbiol 71 (6):3153-7. Hoffman, R. M., D. M. Wolk , S. K. Spencer, and M. A. Borchardt . 2007. Development of a method for the detection of waterborne microsporidia. J Microbiol Methods 70 (2):312-8 .

36. Izq -Mat Izquierdo , F., J. A. Castro Hermida, S. Fenoy , M. Mezo, M. Gonzalez-Warleta , and C. del Aguila . 2011. Detection of microsporidia in drinking water, wastewater and recreational rivers. Water Res 45 (16):4837-43. Keeling, P. 2009. Five questions about microsporidia. PLoS Pathog 5 (9):e1000489. Keeling, P. J., and N. Corradi . 2011. Shrink it or lose it: balancing loss of function with shrinking genomes in the microsporidia. Virulence 2 (1):67-70. Kotler, D. P., and J. M. Orenstein. 1998. Clinical syndromes associated with microsporidiosis . Adv Parasitol 40:321-49. Li, H., F. Qiang , Z. Yang, et al. 2011. Hepatitis B virus with pre-S2 deletion is more prevalent in hepatocellular carcinoma than in chronic active hepatitis and asymptomatic carriers. Acta Virol 55 (2):183-5. Loh , R. S., C. M. Chan, S. E. Ti , L. Lim, K. S. Chan, and D. T. Tan. 2009. Emerging prevalence of microsporidial keratitis in Singapore: epidemiology, clinical features, and management. Ophthalmology 116 (12):2348-53. Mathis, A., R. Weber, and P. Deplazes . 2005. Zoonotic potential of the microsporidia. Clin Microbiol Rev 18 (3):423-45 .

37. Men-Pee Menotti , J., B. Cassinat , C. Sarfati , O. Liguory , F. Derouin , and J. M. Molina. 2003. Development of a real-time PCR assay for quantitative detection of Encephalitozoon intestinalis DNA. J Clin Microbiol 41 (4):1410-3. Mohindra , A. R., M. W. Lee, G. Visvesvara , et al. 2002. Disseminated microsporidiosis in a renal transplant recipient. Transpl Infect Dis 4 (2):102-7. Moretto , M., L. Casciotti , B. Durell , and I. A. Khan. 2000. Lack of CD4(+) T cells does not affect induction of CD8(+) T-cell immunity against Encephalitozoon cuniculi infection. Infect Immun 68 (11):6223-32. Ojuromi , O. T., F. Izquierdo, S. Fenoy , et al. 2012. Identification and characterization of microsporidia from fecal samples of HIV-positive patients from Lagos, Nigeria. PLoS One 7 (4):e35239. Orenstein, J. M., D. T. Dieterich , and D. P. Kotler. 1992. Systemic dissemination by a newly recognized intestinal microsporidia species in AIDS. AIDS 6 (10):1143-50. Peer, C. J., S. D. Spencer, D. A. VanDenBerg , M. A. Pacanowski , R. B. Horenstein , and W. D. Figg . 2012. A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 880 (1):132-9.

38. Pol- Sak Polley , S. D., S. Boadi , J. Watson, A. Curry, and P. L. Chiodini . 2011. Detection and species identification of microsporidial infections using SYBR Green real-time PCR. J Med Microbiol 60 (Pt 4):459-66. Raynaud, L., F. Delbac , V. Broussolle , et al. 1998. Identification of Encephalitozoon intestinalis in travelers with chronic diarrhea by specific PCR amplification. J Clin Microbiol 36 (1):37-40. Robinson, T., S. G. Campino , S. Auburn, et al. 2011. Drug-resistant genotypes and multi- clonality in Plasmodium falciparum analysed by direct genome sequencing from peripheral blood of malaria patients. PLoS One 6 (8):e23204. Sak , B., D. Brady, M. Pelikanova , et al. 2011. Unapparent microsporidial infection among immunocompetent humans in the Czech Republic. J Clin Microbiol 49 (3):1064-70. Sak , B., J. Salat , H. Horka , K. Sakova , and O. Ditrich . 2006. Antibodies enhance the protective effect of CD4+ T lymphocytes in SCID mice perorally infected with Encephalitozoon cuniculi . Parasite Immunol 28 (3):95-9. Sak , B., M. Kvac , Z. Kucerova , D. Kvetonova , and K. Sakova . 2011. Latent microsporidial infection in immunocompetent individuals - a longitudinal study. PLoS Negl Trop Dis 5 (5):e1162 .

39. Sch -Sri Schwartz , D. A., R. T. Bryan, K. O. Hewan -Lowe, et al. 1992. Disseminated microsporidiosis ( Encephalitozoon hellem ) and acquired immunodeficiency syndrome. Autopsy evidence for respiratory acquisition. Arch Pathol Lab Med 116 (6):660-8. Sheth , S. G., C. Bates, M. Federman , and S. Chopra. 1997. Fulminant hepatic failure caused by microsporidial infection in a patient with AIDS. AIDS 11 (4):553-4. Snowden, K. F., E. S. Didier, J. M. Orenstein, and J. A. Shadduck . 1998. Animal models of human microsporidial infections. Lab Anim Sci 48 (6):589-92. Snowden, K. F., K. Logan, and D. N. Phalen . 2000. Isolation and characterization of an avian isolate of Encephalitozoon hellem . Parasitology 121 ( Pt 1):9-14. Sokolova , O. I., A. V. Demyanov , L. C. Bowers, et al. 2011. Emerging microsporidian infections in Russian HIV-infected patients. J Clin Microbiol 49 (6):2102-8. Soule, J. B., A. L. Halverson, R. B. Becker, M. C. Pistole , and J. M. Orenstein. 1997. A patient with acquired immunodeficiency syndrome and untreated Encephalitozoon ( Septata ) intestinalis microsporidiosis leading to small bowel perforation. Response to albendazole . Arch Pathol Lab Med 121 (8):880-7. Sridhar, M. S., and S. Sharma. 2003. Microsporidial keratoconjunctivitis in a HIV-seronegative patient treated with debridement and oral itraconazole . Am J Ophthalmol 136 (4):745-6 .

40. Tal-Web Talabani , H., C. Sarfati , E. Pillebout , T. van Gool , F. Derouin , and J. Menotti. 2010. Disseminated infection with a new genovar of Encephalitozoon cuniculi in a renal transplant recipient. J Clin Microbiol 48 (7):2651-3. Terada, S., K. R. Reddy, L. J. Jeffers, A. Cali, and E. R. Schiff. 1987. Microsporidan hepatitis in the acquired immunodeficiency syndrome. Ann Intern Med 107 (1):61-2. Torres, G., F. Izquierdo, V. Capo, et al. 2012. Genital microsporidiosis in women with AIDS: A post-mortem study. Rev Iberoam Micol . U.S.E.P.A. 2005. Method 1623: Cryptosporidium and Giardia in water by filtration/IMA/FA. Vávra , J. and Larson, J. I. R. 1999. Structure of the microsporidia. In The microsporidia and microsporidiosis ed. M, Witter and L.M. Wittner , 7-84. Washintong D.C. : ASM Press Velasquez , J. N., A. V. Chertcoff , C. Etchart , et al. 2012. First case report of infection caused by Encephalitozoon intestinalis in a domestic cat and a patient with AIDS. Vet Parasitol . Visvesvara , G. S., and L. S. Garcia. 2002. Culture of protozoan parasites. Clin Microbiol Rev 15 (3):327-8. Weber, R., R. T. Bryan, D. A. Schwartz, and R. L. Owen. 1994. Human microsporidial infections. Clin Microbiol Rev 7 (4):426-61 .

41. Web- Wic Weber , R., R. T. Bryan, D. A. Schwartz, and R. L. Owen. 1994. Human microsporidial infections. Clin Microbiol Rev 7 (4):426-61. Weber, R., R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin , and G. S. Visvesvara . 1992. Improved light- microscopical detection of microsporidia spores in stool and duodenal aspirates. The Enteric Opportunistic Infections Working Group. N Engl J Med 326 (3):161-6. Wichro , E., D. Hoelzl , R. Krause, G. Bertha, F. Reinthaler , and C. Wenisch . 2005. Microsporidiosis in travel-associated chronic diarrhea in immune-competent patients. Am J Trop Med Hyg 73 (2):285-7.