[Ejaculation capacity in spinal cord injured patients carrying an endo-urethral stent for incontinentation: Descriptive study]. / Capacité éjaculatoire chez les sujets blessés médullaires porteurs d'une prothèse endo-urétrale d'incontinentation : étude descriptive.

OBJECTIVES: To assess the impact of endo-urethral stent (EUP) of incontinentation in spinal cord injured (SCI) men on ejaculation capacity. FIRST CRITERIA: Presence of ejaculation after EUP. SECONDARY CRITERIA: Modification on ejaculation type, stimulation mode, possibility of sperm conservation in the same population. POPULATION AND METHODS: A descriptive monocentric study including SCI subjects which underwent placement or change of temporary (Mémokath™ or Allium™) or definitive (Mémotherm™) EUP between January 2004 and January 2014 with at least one ejaculatory test. Are identified for each patient: the presence of ejaculation, type of ejaculation, stimulation mode used, number of frozen specimen and results from semen analysis. RESULTS: Over 11 men with tetraplegia, complete for motricity, there were 8 (72%) for whom ejaculation was possible after laying EUP. Of these, 6 (55%) had an antegrade or antero-retrograde ejaculation. It was not possible to make a freezing straw in four of them due to infection of semen (2) or terato-oligo-astenospermia (1) or absence of reproductive project (1). CONCLUSION: In this small sample, it was found that over 70% ejaculation have been preserved after laying EUP and more than 50% with antegrade or antero-retrograde ejaculation. A larger study would identify how EUP may alter the ejaculatory capacity or quality of the ejaculate. LEVEL OF EVIDENCE: 4.

Vectorial role of Acanthamoeba in Legionella propagation in water for human use.

Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown.

Novel Acanthamoeba 18S rRNA gene sequence type from an environmental isolate.

The free-living amoebae, Acanthamoeba, can act as opportunistic parasites on a wide range of vertebrates and are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices, and their ubiquitous distribution. Subgenus classification based on morphology is being replaced by a classification based on the sequences of the 18S rRNA gene with a total of 18 different genotypes (T1-T18). A new environmental strain of Acanthamoeba isolated from a waste water treatment plant is presented in this study as a candidate for the description of the novel genotype T19 after phylogenetic analysis.

A year-long study of Cryptosporidium species and subtypes in recreational, drinking and wastewater from the central area of Spain.

A year-long longitudinal study was undertaken to evaluate the presence of Cryptosporidium spp. in drinking water treatment plants (DWTPs), wastewater treatment plants (WWTPs) and freshwater bathing beaches (FBBs) from the central area of Spain. Water samples were collected according to USEPA Method 1623, and concentrated by the IDEXX Filta-Max® system. Cryptosporidium species were detected based on PCR-restriction fragment length polymorphism and sequence analyses of the ssuRNA gene. C. hominis and/or C. parvum isolates were subtyped by DNA sequencing of the Gp60 gene. Among 150 samples, 23 (15.3%) were positive by IFAT and 40 (26.7%) by PCR. Cryptosporidium spp. was more frequent in WWTPs (26.2 and 50.8%) and FBBs (12.5 and 17.5%) by IFAT and PCR respectively. Effluent waters from DWTPs were negative for this parasite suggesting that they are suitable for public use. Tertiary treatment in the WWTPs demonstrated a high removal efficiency of Cryptosporidium in the samples evaluated. Cryptosporidium species identified included C. hominis, C. parvum, C. ubiquitum, C. andersoni and C. muris. Subtyping analysis revealed C. hominis IbA10G2 and IeA11G3T3 alleles, which is the first report of the latter in water samples. Cryptosporidium highest frequency was observed in winter and spring. Our data provide information about the occurrence and diversity of Cryptosporidium in water of human use from the central area of Spain.

A year long study of the presence of free living amoeba in Spain.

Free-living amoeba such as Acanthamoeba and Balamuthia mandrillaris can act as opportunistic parasites on a wide range of vertebrates and they are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices and their ubiquitous distribution. This work was carried out in order to study the presence of these free-living amoebae (FLA) and their possible seasonality in a continental-Mediterranean climate in different types of water. For this purpose, a total of 223 water samples were collected during one year from four drinking water treatment plants (DWTP), seven wastewater treatment plants (WWTP) and six locations of influence (LI) on four river basins from Spain. Water samples were concentrated using the IDEXX Filta-Max(®) system and analyzed by a triplex real time PCR that detects Acanthamoeba, B. mandrillaris and Naegleria fowleri. Agar plates were also seeded for Acanthamoeba culture. From the three FLA studied, N. fowleri was not detected in any sample while B. mandrillaris was found at the entrance of a DWTP; this being, to our knowledge, the first report of these protozoa in water worldwide. On the other hand, the presence of Acanthamoeba observed was higher, 94.6% of the studied points were positive by real time PCR and 85.2% by culture, resulting in 99.1% positive for Acanthamoeba with both methods. All genetically analyzed Acanthamoeba were genotype T4 but nine different T4/DF3 sequences were observed, three of them being described for the first time, assigning new codes. No seasonal distribution of Acanthamoeba was found. These facts should serve as a warning to contact lens wearers of the risk of a poor hygiene when handling their contact lenses. It should also serve as a signal to physicians to consider FLA as a possible causative agent of nervous system infections as well as Acanthamoeba keratitis due to their high environmental presence shown in this study.

Molecular characterization of Acanthamoeba isolated in water treatment plants and comparison with clinical isolates.

A total of 116 samples (44 clinical specimens and 72 environmental samples) have been analyzed for the presence of Acanthamoeba. The environmental samples (ESs) were collected from four drinking water treatment plants (DWTP, n=32), seven wastewater treatment plants (n=28), and six locations of influence (n=12) on four river basins from the central area of Spain (winter-spring 2008). Water samples were concentrated by using the IDEXX Filta-Max(®) system. Acanthamoeba was identified in 65 of the 72 ESs by culture isolation (90.3%) and 63 by real-time PCR (87.5%), resulting in all sampling points (100%) positive for Acanthamoeba when considering both techniques and all the time period analyzed. Nine of the 44 clinical specimens were positive for Acanthamoeba. Seventeen Acanthamoeba strains (eight from four DWTP and nine from clinical samples) were also established in axenic-PYG medium. Twenty-four of the ESs and the 17 Acanthamoeba sp. strains were genotyped as T4/1, T4/8, and T4/9. The eight strains isolated from the DWTP samples were inoculated in nude mouse to ascertain their potential pathogenicity in this model. Animals that were inoculated died or showed central nervous system symptoms 9 days post-inoculation. Examination of immunofluorescence-stained brain and lung tissue sections showed multiple organisms invading both tissues, and re-isolation of throphozoites was successful in these tissues of all infected animals. For the first time, potentially pathogenic Acanthamoeba T4 has been detected in 100% of different types of water samples including tap water and sewage effluents in the central area of Spain suggesting a potential health threat for humans especially for the contact lens wearers.

Detection of zoonotic intestinal parasites in public parks of Spain. Potential epidemiological role of microsporidia.

Several studies have demonstrated that the soil of public parks presents an important source of infection which has a significant impact on public health. Children are the main group affected by accidentally ingestion of contaminated soil. This study was performed in order to identify the presence of zoonotic parasites in dog and cat faecal and soil samples from public parks of Madrid, Spain. Six hundred twenty-five and seventy-nine soil and faecal samples (presumably from dogs and cats) respectively were collected from 67 parks. Intestinal parasites were identified in 27 parks (40.3%), which were contamined with Giardia sp. (19.4%), microsporidia (19.4%), Toxocara spp. (16.4%), Cryptosporidium sp. (6%), Entamoeba histolytica (3%) and Ancylostomidae (3%). Combinations of two or more intestinal parasites were found in 11 parks, and it was common to find Giardia and microsporidia together in samples. Intestinal parasites were detected in 18% (112/625) of soil samples. The most frequent parasite species found in the examined soil samples were Toxocara spp. (16.4%), followed by Giardia sp. (4.5%) and Strongyloides sp. larvae (3%). The zoonotic parasites found in the 79 faecal samples were Giardia sp. (17.7%), Cryptosporidium sp. (9%), E. histolytica (2.5%), Trichuris vulpis (1.3%), Toxascaris leonina (1.3%) and microsporidia spores (28%). Microsporidia characterization by amplification of DNA confirmed 10 samples as positive, eight for E. bieneusi and two for E. hellem by PCR. The role of those parasites in the environment are discussed.

First cases of microsporidiosis in transplant recipients in Spain and review of the literature.

Microsporidia are currently considered emerging pathogens responsible for life-threatening infections in organ transplant recipients. Here, we describe the first cases of intestinal microsporidiosis by Enterocytozoon bieneusi genotype D in two non-HIV-infected renal transplant recipients from Spain. Previously reported cases of microsporidiosis in organ transplant recipients have also been reviewed, highlighting the necessity of considering organ transplant recipients a risk group for microsporidiosis. A systematic search for these parasites is recommended in cases of persistent diarrhea and in the differential diagnosis of other syndromes, such as chronic fever of unknown etiology.

High-level resistance of Nosema ceranae, a parasite of the honeybee, to temperature and desiccation.

Resistance of Nosema ceranae to different exposure conditions has been evaluated by using Sytox green and DAPI (4',6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35 degrees C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4 degrees C were also detected.

Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected cells.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

First detection and genotyping of human-associated microsporidia in pigeons from urban parks.

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.

Enterocytozoon bieneusi (microsporidia) in faecal samples from domestic animals from Galicia, Spain.

In this survey we examined 87 domestic animal stool samples in order to detect the possible presence of microsporidia in animals in close contact with humans in Galicia (NW, Spain). The detection of Enterocytozoon bieneusi spores was confirmed in faecal samples from two dogs and one goat by polymerase chain reaction. None of the positive samples for microsporidia in the staining method were amplified with species-specific primers for Encephalitozoon intestinalis, E. hellem and E. cuniculi. Four rabbits faecal samples reacted with anti-E. cuniculi serum. Our results could indicate the importance of domestic animals as zoonotic reservoirs of microsporidial human infections.

Animal models in ocular toxocariasis.

Ocular toxocariasis is a clearly defined disease. However, much remains to be learned concerning the migratory route, ocular changes, diagnosis and treatment. Studies in paratenic hosts have contributed to our understanding and will yield more information. Various experimental animals have been used, such as mice, rabbits, guinea pigs, primates, hamsters and gerbils. Of these, the last appear to be the most appropriate model due to their high susceptibility to ocular infection. Results obtained from different animal models are often not comparable due to the fact that dose and routes of inoculation are diverse. Early stages in the pathogenesis of ocular toxocariasis are manifested by haemorrhages in the anterior chamber and iris, replaced in time by accumulations of white cells. Ocular migration produces an early cell reaction, formed by an infiltration of neutrophils accompanied by vasculitis and retinal microinfarcts. Over a period of time, an increase of macrophages and the distribution of the infiltrates is observed. Later, granulomatous lesions are formed. These do not necessarily contain a larva and their appearance varies in different animal models. Local production of IgE and the presence of specific IgG have been described.

Isotype specific immune responses in murine experimental toxocariasis.

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.

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

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

Microsporidiosis in travelers with diarrhea from the tropics.

BACKGROUND: Microsporidia are protozoa which mainly affect severely immunodepressed AIDS patients in developed countries as well as those in developing ones. Traveler's diarrhea affects approximately 40% of people traveling from industrialized countries to developing ones, and no pathogens are identified in many of those patients on their returning, suggesting that known enteropathogens escape detection or entirely new ones could be responsible. Very few reports of travel-related microsporidiosis have been described. METHODS: Between January, 1996 and January, 1998, a total of 40 European travelers from the tropics with a clinical picture of protected diarrhea (three or more loose stools per day lasting for more than 3 weeks) were evaluated. Weber's trichrome modified by Kokoskin stain for microsporidial spores were performed in stool samples of every patient. Microsporidial DNA extraction and PCR amplification were attempted in every stool sample where microsporidial spores were observed. RESULTS: Four cases of imported Enterocytozoon bieneusi were detected: one HIV-infected short term traveler, a pregnant long term traveler, and two immunocompetent short term travelers. Diarrhea was self-limited, and the spores cleared from the stools in all HIV-non infected travelers, but showed a chronic course in the HIV-infected one. CONCLUSIONS: Available data is too limited to affirm that residence or travel in tropical countries increases the risk for microsporidial infection, but the cases presented here suggest that E. bieneusi could be a cause of self-limited diarrhea in immunocompetent travelers returning from the tropics or could chronically affect immunocompromised HIV-infected travelers.