Carmen del Aguila

Intestinal microsporidiosis due to Enterocytozoon bieneusi in elderly human immunodeficiency virus-negative patients from Vigo, Spain

We report what is, to our knowledge, the first study in which microsporidial infection was detected in elderly human immunodeficiency virus (HIV)--negative patients. Of the 60 elderly patients studied, 47 had diarrhea. Intestinal microsporidiosis due to Enterocytozoon bieneusi was diagnosed in 8 patients (17.02%) by use of Webers chromotrope-based stain and polymerase chain reaction with species-specific primers. The mean age of these 8 patients was 75 years; 7 had chronic diarrhea and 1 had nonchronic diarrhea. Six of the patients with chronic diarrhea had no other pathogens isolated. In our opinion, elderly patients, because of their special immunological characteristics, should be considered a group at risk for the acquisition of intestinal microsporidiosis.

Phylogenetic Approach to the Variability of the Microsporidian Enterocytozoon bieneusi and Its Implications for Inter- and Intrahost Transmission

ABSTRACT Enterocytozoon bieneusi is a microsporidian parasite that infects many vertebrate animals, including humans. The rDNA internal transcribed spacer (ITS) shows a hypervariable sequence; however, so far no clear information has been inferred about strain evolution in this species. We reviewed all the sequences described and performed a phylogenetic study. Four groups of sequences strongly differentiated from each other were detected, although most of the isolates (94%) corresponded to group I. The highly diverse sequences of this group were analyzed using median-joining networks. The host species (humans, pets, swine, cattle, birds, and wild animals) and the continents of origin of the isolates were considered. Central haplotypes in the network were obtained from very diverse hosts and geographical origins. The results show that although E. bieneusi has a broad host specificity, transmission is not completely free: some strains were able to circulate within a given host species and were only occasionally transmitted to another host. Additionally, while not relevant for swine or cattle hosts, geography seems to be a relevant factor for human infection by E. bieneusi.

Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees.

Nosema ceranae is currently one of the major pathogens of honeybees, related to the worldwide colony losses phenomenon. The genotyping of strains based on ribosomal DNA (rDNA) can be misleading if the repeated units are not identical. The analysis of cloned rDNA fragments containing the intergenic spacer (IGS) and part of the rDNA small-subunit (SSU) gene, from N. ceranae isolates from different European and Central Asia populations, revealed a high diversity of sequences. The variability involved single-nucleotide polymorphisms and insertion/deletions, resulting in 79 different haplotypes. Two sequences from the same isolate could be as different as any pair of sequences from different samples; in contrast, identical haplotypes were also found in very different geographical origins. Consequently, haplotypes cannot be organized in a consistent phylogenetic tree, clearly indicating that rDNA is not a reliable marker for the differentiation of N. ceranae strains. The results indicate that recombination between different sequences may produce new variants, which is quite surprising in microsporidia, usually considered to have an asexual mode of reproduction. The diversity of sequences and their geographical distribution indicate that haplotypes of different lineages may occasionally be present in a same cell and undergo homologue recombination, therefore suggesting a sexual haplo-diploid cycle.

Intraspecies Genotype Variability of the Microsporidian Parasite Encephalitozoon hellem

ABSTRACT Seven isolates of Encephalitozoon hellem from human immunodeficiency virus-positive patients were genotyped through a series of markers: the internal transcribed spacer (ITS) of ribosomal DNA, the polar tube protein (PTP) gene, and two intergenic spacers (IGS-TH and IGS-HZ) whose polymorphism is newly reported. The genome markers were all analyzed at three levels: PCR amplification followed by polyacrylamide gel electrophoresis, single-strand conformation analysis (SSCA), and DNA sequencing. The polymorphisms detected involve insertions/deletions and point mutations. SSCA can distinguish any pair of sequences, even those differing by a single base pair. The different isolates studied fit into the previously described ITS genotype 1A, except one which seems to be a 2A derivative variant (2D). When PTP and the new markers IGS-TH and IGS-HZ were analyzed, most of the isolates displayed different genotypes, demonstrating that E. hellem has a strong intraspecies variability. A set of markers such as those used here may be very useful in genotyping of clinical samples and in the assessment of epidemiological relationships among E. hellem strains.

Detection of Enterocytozoon bieneusi in Two Human Immunodeficiency Virus—Negative Patients with Chronic Diarrhea by Polymerase Chain Reaction in Duodenal Biopsy Specimens and Review

Intestinal microsporidiosis has been associated traditionally with severely immunocompromised patients with AIDS. We describe two new cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in human immunodeficiency virus-negative adults. Both patients presented with chronic nonbloody diarrhea, and one had intestinal lymphangiectasia as well. Intestinal microsporidiosis was diagnosed by evaluation of stool samples, and the specific species was determined by use of polymerase chain reaction (PCR) in duodenal biopsy specimens. To our knowledge, this is the first report of confirmation of E. bieneusi in the intestinal epithelium of HIV-negative individuals by use of PCR in duodenal biopsy specimens. Cases of intestinal microsporidiosis in HIV-negative individuals reported in the English-language literature are reviewed. These two new cases along with those described previously corroborate the need to evaluate for microsporidia in HIV-negative individuals with unexplained diarrhea.

Molecular Characterization of Human-Pathogenic Microsporidia and Cyclospora cayetanensis Isolated from Various Water Sources in Spain: a Year-Long Longitudinal Study

ABSTRACT Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis, Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae. C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.

In Vitro Culture, Ultrastructure, Antigenic, and Molecular Characterization of Encephalitozoon cuniculi Isolated from Urine and Sputum Samples from a Spanish Patient with AIDS

ABSTRACT 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.

Cryptosporidium: un género en revisión. Situación en España

Genus Cryptosporidium, has undergone major revisions in recent years. The identification of new species and their major reservoirs has contributed to the knowledge of the epidemiology of human infection. In Spain, although there are many publications, few studies have been conducted to identify the circulating species and genotypes. This fact has led us to review and update these new studies published in Spain, particularly those that use molecular methods in order to characterise the species and genotypes present in our country.

Detection and genotyping of Enterocytozoon bieneusi in pigeons

TO date, eight genera of microsporidia are known to infect humans. These obligate intracellular parasites are able to proliferate in all animal phyla. However, the epidemiology of these parasites and their transmission routes are not well understood. Microsporidiosis is not included or diagnosed in most routine clinical analysis protocols, and all potential host species are not known (del Aguila et al. 1999; Dengjel et al. 2001; Haro et al. 2005). Thus, the zoonotic origin of human microsporidiosis is far from clear and the epidemiology of different microsporidia species differ (Mathis, Weber, and Deplazes 2005). Encephalitozoon hellem is mainly found in avian hosts (Mathis et al. 2005) and rarely in humans (Didier et al. 2004). Enterocytozoon bieneusi is the most prevalent microsporidia in humans (Didier et al. 2004) but it has also been found in a number of different animal species including racoons, dogs, and pigs (Mathis et al. 2005; Sulaiman et al. 2003). Recently, waterborne transmission has been recognized as a potential source of microsporidial infection (Cotte et al. 1999; Franzen and Muller 1999) and aquatic birds have been implicated in this mode of transmission (Slodkowicz-Kowalska et al. 2006). As emerging parasites, it is important not only to identify microsporidia at the species level, but also to assign specific genotypes that can assess theoretical epidemiological correlations between strains, hosts, and the environment. The Internal transcribed spacer (ITS between the small and the large ribosomal subunit of RNA genes) region is the most frequently used sequence to determine genetic diversity between strains (Haro et al. 2003; Mathis et al. 2005). The E. hellem ITS genotype A1 is most frequently found in HIV-positive patients from Europe and the USA (Haro et al. 2003), and we recently detected it in a Spanish park-pigeon isolate. This suggests that transmission of E. hellem can occur between park-pigeons and humans (Haro et al. 2005). In that earlier study we identified several species of microsporidia in public park pigeons (Columba livia) in Spain, including E. bieneusi, and we here report on the ITS variability of E. bienusi.

Microsporidiosis in HIV‐Positive Children in Madrid (Spain).

A prospective study was carried out to determine the prevalence rates of microsporidiosis and other enteroparasites in HIV‐positive children in the Madrid area. HIV‐positive pediatric patients from three hospitals were entrolled in the study. A total of 293 samples (158 stool and 127 urine) were collected from 83 children whose mean age was 6.3 years and hed a mean CD4 count of 504.7/mm3 (range 1‐2,220/mm3), 48 of whom suffered diarrhea at the time of the study. Microsporidia indentification was investigated in stool and unrine samples using Webers chromotrope‐based strain, IIF and PCR species‐specific tests. Enteric parasites were identified in 32.5% of the children. Cryptosporidium sp. was the most common parasite encuntered (14.4%), followed by Blastocytis sp. (9.6%) and Giardia duodenalis (8.4%). Microsoridia was only found in the stools of one child (1.2% of total and 2% of those with diarrhea) and Enterocytozoon bieneusi was demonstrated by PCR. The patient was 10 years old, Presented non‐chronic diarrhea and his CD4 count was 298/mm3. These date differ from those previously reported by us in HIV‐positive adults (13.9%) in the same area, although this group showed more severely depressed CD4 lymphocyte counts than children. New epidemiological studies should be carried out to elucidate whether additional risk factors exist betwen these groups.