PCR Amplification and Species Determination of Microsporidia in Formalin-Fixed Feces after Immunomagnetic Separation

The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.     

Intestinal microsporidiosis in India: A two year study

Intestinal parasitic pathogens in HIV/AIDS patients include Cryptosporidium sp, Cystoisospora sp, microsporidia and less commonly other parasites. The two most common microsporidia causing intestinal infection are Enterocytozoon bieneusi and Encephalitozoon intestinalis. Most of the Indian studies for intestinal parasitic infections in HIV/AIDS patients have not included microsporidia, due to difficult staining and identification of the parasite. The aim of the present study was to find the prevalence of intestinal microsporidiosis and their species identification along with correlation of CD4 count with parasite positivity and diarrhoea in HIV positive individuals. Stool samples of 363 individuals including 125 HIV seropositive patients with diarrhoea, 158 HIV seropositive patients without diarrhoea, 55 HIV seronegative patients with diarrhoea and 25 healthy controls were obtained from various out-patient departments and in-patients admitted to a tertiary care hospital from August 2008 to October 2009. The stool samples were subjected to examination by wet mount, modified acid fast stain for coccidian parasites and multiplex nested PCR for microsporidia. The overall prevalence of all intestinal parasites among HIV patients in our study was 26.5%. The prevalence of intestinal parasitic pathogens in HIV positive patients with diarrhoea was 43.2%. Microsporidia were the most common parasites detected (14%) in all patients, while in HIV infected patients 15.9% patients had microsporidia infection. The most common species causing intestinal microsporidiosis in our study was E. intestinalis (10.5%). In HIV seropositive individuals with diarrhoea, E. intestinalis was 20.8% and E. bieneusi 8.0% while in HIV-seropositive individuals without diarrhoea, E. intestinalis was 3.8% and E. bieneusi 1.9%. E. intestinalis was present in 10.9% of HIV negative individuals with diarrhoea in whom E. bieneusi was not found. There was a significant association between CD4 count ≤ 200/μl and intestinal parasite positivity. Thus, it can be concluded that intestinal microsporidiosis is under reported but an important disease in India. The predominant species in our study is E. intestinalis , in contrast to other parts of the world where E. bieneusi is more common.     

Frequent Occurrence of Human-Associated Microsporidia in Fecal Droppings of Urban Pigeons in Amsterdam, The Netherlands

Human-associated microsporidia were frequently observed in fecal samples of 331 feral pigeons in Amsterdam, The Netherlands, obtained during high- and low-breeding periods. Thirty-six of 331 samples (11%) contained the human pathogens Enterocytozoon bieneusi (n = 18), Encephalitozoon hellem (n = 11), Encephalitozoon cuniculi (n = 6), and Encephalitozoon intestinalis (n = 1); 5 samples contained other microsporidia. Pigeon feces can be an important source of human microsporidian infection.     

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

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.     

Identification of Encephalitozoon cuniculi genotype III and two novel genotypes of Enterocytozoon bieneusi in swine

Samples of intestinal content from thirty fattened pigs of six farms slaughtered at an abattoir in North-Western Germany, and faecal samples of four pigs kept as laboratory animals at the Federal Institute for Risk Assessment (BfR, Berlin, Germany) were investigated for the occurrence of microsporidia by light microscopy, PCR and sequencing. A modified Webers trichrome staining and the immunohistochemistry (the Avidin-Biotin-Peroxidase-Complex technique with a polyclonal anti-Encephalitozoon cuniculi-serum and monoclonal antibodies against Encephalitozoon intestinalis and Enterocytozoon bieneusi) was used as a screening method for the light microscopical detection of these pathogenic eukaryotes. By this light microscopically methods microsporidia suspected organisms were found in all samples (100%). By the use of PCR, microsporidia were identified in fourteen samples (41.2%). The prevalence of microsporidia infections among the farms diversifies from 0 to 80% as considered by PCR. E. bieneusi was the most prevalent species and was identified in twelve fattened pigs (40%) from five of the six tested farms (83.3%) and in two of the four laboratory animals (50%). Three of the E. bieneusi species belonged to the genotype O, one to the genotype E, and one to the genotype F. Two isolates were identified as novel genotypes and two samples showed a mixed infection of different genotypes. In three faecal samples of the pigs from two farms E. cuniculi genotype III was identified. One sample contained both microsporidia species. To our knowledge, this is the first time that the genotype III of E. cuniculi was identified in swine.     

Quantitative Assessment of Contamination of Fresh Food Produce of Various Retail Types by Human-Virulent Microsporidian Spores

This study demonstrated that fresh food produce, such as berries, sprouts, and green-leafed vegetables, sold at the retail level can contain potentially viable microsporidian spores of human-virulent species, such as Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon cuniculi, at quantities representing a threat of food-borne infection.     

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.     

Identification and characterization of microsporidia from fecal samples of HIV-positive patients from Lagos, Nigeria

Background

Microsporidia are obligate intracellular parasites that infect a broad range of vertebrates and invertebrates. They have been increasingly recognized as human pathogens in AIDS patients, mainly associated with a life-threatening chronic diarrhea and systemic disease. However, to date the global epidemiology of human microsporidiosis is poorly understood, and recent data suggest that the incidence of these pathogens is much higher than previously reported and may represent a neglected etiological agent of more common diseases indeed in immunocompetent individuals. To contribute to the knowledge of microsporidia molecular epidemiology in HIV-positive patients in Nigeria, the authors tested stool samples proceeding from patients with and without diarrhea.

Methodology/Principal Findings

Stool samples from 193 HIV-positive patients with and without diarrhea (67 and 126 respectively) from Lagos (Nigeria) were investigated for the presence of microsporidia and Cryptosporidium using Weber’s Chromotrope-based stain, Kinyoun stain, IFAT and PCR. The Weber stain showed 45 fecal samples (23.3%) with characteristic microsporidia spores, and a significant association of microsporidia with diarrhea was observed (O.R.  = 18.2; CI: 95%). A similar result was obtained using Kinyoun stain, showing 44 (31,8%) positive samples with structures morphologically compatible with Cryptosporidium sp, 14 (31.8%) of them with infection mixed with microsporidia. The characterization of microsporidia species by IFAT and PCR allowed identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis and E. cuniculi in 5, 2 and 1 samples respectively. The partial sequencing of the ITS region of the rRNA genes showed that the three isolates of E.bieneusi studied are included in Group I, one of which bears the genotype B.

Conclusions/Significance

To our knowledge, this is the first report of microsporidia characterization in fecal samples from HIV-positive patients from Lagos, Nigeria. These results focus attention on the need to include microsporidial diagnosis in the management of HIV/AIDS infection in Nigeria, at the very least when other more common pathogens have not been detected.     

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with “state‐of‐the‐art” equipment and well‐trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore‐concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN‐1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.     

Survey for Zoonotic Microsporidian Pathogens in Wild Living Urban Rooks (Corvus frugilegus)

Microsporidia are opportunistic pathogens in nature infecting all animal phyla. There is a potential risk of microsporidian spores transmission from urban rooks inhabiting some metropolitan cities to people through casual interactions. The aim of this study was to identify microsporidia species in the droppings of rooks in Wroclaw, Poland. A total of 15 collective sets of droppings were examined using nested‐PCR method. Amplification of ITS rRNA gene revealed the presence of Enterocytozoon bieneusi D, Peru 6, and Encephalitozoon hellem 1A genotypes. This study indicates that excreta of urban rooks can be an important source of human infection with these pathogens.     

Molecular Characterization of Microsporidia Indicates that Wild Mammals Harbor Host-Adapted Enterocytozoon spp. as well as Human-Pathogenic Enterocytozoon bieneusi

Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E. bieneusi. However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E. bieneusi and have no apparent public health significance. This is the first report of E. bieneusi in wildlife.     

Identification of a New Microsporidian Parasite Related to Vittaforma corneae in HIV-Positive and HIV-Negative Patients from Portugal

ABSTRACT. Fecal samples from 22 HIV-positive and 3 HIV-negative patients from Portugal with symptomatic diarrhea were diagnosed positive for microsporidia by microscopy, with most parasites detected significantly bigger than Enterocytozoon bieneusi and Encephalitozoon spp. Sequence characterization of the small subunit (SSU) rRNA gene identified a microsporidian parasite with 96% homology to two published Vittaforma corneae sequences. Phylogenetic analysis confirmed the genetic relatedness of this new microsporidian parasite to Vittaforma corneae as well as Cystosporogenes operophterae. Results of the study demonstrate the presence of a new human-pathogenic microsporidian species, which is responsible for significant number of infections in HIV-positive and HIV-negative patients in Portugal.     

Genomic Survey of the Non-Cultivatable Opportunistic Human Pathogen,Enterocytozoon bieneusi

Enterocytozoon bieneusi is the most common microsporidian associated with human disease, particularly in the immunocompromised population. In the setting of HIV infection, it is associated with diarrhea and wasting syndrome. Like all microsporidia, E. bieneusi is an obligate, intracellular parasite, but unlike others, it is in direct contact with the host cell cytoplasm. Studies of E. bieneusi have been greatly limited due to the absence of genomic data and lack of a robust cultivation system. Here, we present the first large-scale genomic dataset for E. bieneusi. Approximately 3.86 Mb of unique sequence was generated by paired end Sanger sequencing, representing about 64% of the estimated 6 Mb genome. A total of 3,804 genes were identified in E. bieneusi, of which 1,702 encode proteins with assigned functions. Of these, 653 are homologs of Encephalitozoon cuniculi proteins. Only one E. bieneusi protein with assigned function had no E. cuniculi homolog. The shared proteins were, in general, evenly distributed among the functional categories, with the exception of a dearth of genes encoding proteins associated with pathways for fatty acid and core carbon metabolism. Short intergenic regions, high gene density, and shortened protein-coding sequences were observed in the E. bieneusi genome, all traits consistent with genomic compaction. Our findings suggest that E. bieneusi is a likely model for extreme genome reduction and host dependence.     

Identification of Potentially Human-Pathogenic Enterocytozoon bieneusi Genotypes in Various Birds

Enterocytozoon bieneusi was detected in 24 of 83 samples from birds of the orders Columbiformes, Passeriformes, and Psittaciformes. It was identical to or closely related to the Peru6 genotype, which was previously found in humans in Peru. Thus, various birds can be a significant source of environmental contamination by potentially human-pathogenic E. bieneusi.     

Comparative Analysis of the Proteins with Tandem Repeats from 8 Microsporidia and Characterization of a Novel Endospore Wall Protein Colocalizing with Polar Tube from Nosema bombycis

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal‐infecting microsporidia (Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect‐infecting microsporidia (Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect‐infecting microsporidia was larger than that of mammal‐infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC‐rich codons. The tandem repeat position within proteins of insect‐infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal‐infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite.     

Prevalence of Cryptosporidium spp., Enterocytozoon bieneusi,Encephalitozoon spp. and Giardia intestinalis in Wild,Semi-Wild and Captive Orangutans (Pongo abelii and Pongo pygmaeus) on Sumatra and Borneo,Indonesia

Background

Orangutans are critically endangered primarily due to loss and fragmentation of their natural habitat. This could bring them into closer contact with humans and increase the risk of zoonotic pathogen transmission.

Aims

To describe the prevalence and diversity of Cryptosporidium spp., microsporidia and Giardia intestinalis in orangutans at seven sites on Sumatra and Kalimantan, and to evaluate the impact of orangutans’ habituation and location on the occurrence of these zoonotic protists.

Result

The overall prevalence of parasites in 298 examined animals was 11.1%. The most prevalent microsporidia was Encephalitozoon cuniculi genotype II, found in 21 animals (7.0%). Enterocytozoon bieneusi genotype D (n = 5) and novel genotype Pongo 2 were detected only in six individuals (2.0%). To the best of our knowledge, this is the first report of these parasites in orangutans. Eight animals were positive for Cryptosporidium spp. (2.7%), including C. parvum (n = 2) and C. muris (n = 6). Giardia intestinalis assemblage B, subtype MB6, was identified in a single individual. While no significant differences between the different human contact level groups (p = 0.479–0.670) or between the different islands (p = 0.992) were reported in case of E. bieneusi or E. cuniculi, Cryptosporidium spp. was significantly less frequently detected in wild individuals (p < 2×10⁻¹⁶) and was significantly more prevalent in orangutans on Kalimantan than on Sumatra (p < 2×10⁻¹⁶).

Conclusion

Our results revealed that wild orangutans are significantly less frequently infected by Cryptosporidium spp. than captive and semi-wild animals. In addition, this parasite was more frequently detected at localities on Kalimantan. In contrast, we did not detect any significant difference in the prevalence of microsporidia between the studied groups of animals. The sources and transmission modes of infections were not determined, as this would require repeated sampling of individuals, examination of water sources, and sampling of humans and animals sharing the habitat with orangutans.     

Recovery, Bioaccumulation, and Inactivation of Human Waterborne Pathogens by the Chesapeake Bay Nonnative Oyster, Crassostrea ariakensis

The introduction of nonnative oysters (i.e., Crassostrea ariakensis) into the Chesapeake Bay has been proposed as necessary for the restoration of the oyster industry; however, nothing is known about the public health risks related to contamination of these oysters with human pathogens. Commercial market-size C. ariakensis triploids were maintained in large marine tanks with water of low (8-ppt), medium (12-ppt), and high (20-ppt) salinities spiked with 1.0 × 10⁵ transmissive stages of the following human pathogens: Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores (i.e., Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi). Viable oocysts and spores were still detected in oysters on day 33 post-water inoculation (pwi), and cysts were detected on day 14 pwi. The recovery, bioaccumulation, depuration, and inactivation rates of human waterborne pathogens by C. ariakensis triploids were driven by salinity and were optimal in medium- and high-salinity water. The concentration of human pathogens from ambient water by C. ariakensis and the retention of these pathogens without (or with minimal) inactivation and a very low depuration rate provide evidence that these oysters may present a public health threat upon entering the human food chain, if harvested from polluted water. This conclusion is reinforced by the concentration of waterborne pathogens used in the present study, which was representative of levels of infectious agents in surface waters, including the Chesapeake Bay. Aquacultures of nonnative oysters in the Chesapeake Bay will provide excellent ecological services in regard to efficient cleaning of human-infectious agents from the estuarine waters.     

Genotyping of Enterocytozoon bieneusi in Farmed Blue Foxes (Alopex lagopus) and Raccoon Dogs (Nyctereutes procyonoides) in China

Enterocytozoon bieneusi is the most common species of microsporidia found both in humans and animals. Farmed animals, particularly closely associated to humans, may play an important role of zoonotic reservoir in transmitting this disease to humans. The fur industry is a major economic component in some parts of China. To understand the prevalence, genotype variety and zoonotic risk of E. bieneusi in farmed foxes and raccoon dogs, two species of fur animals, fecal specimens of 110 blue foxes and 49 raccoon dogs from Heilongjiang and Jilin provinces in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 16.4% (18/110) blue foxes and 4.1% (2/49) raccoon dogs. Altogether, four genotypes of E. bieneusi were identified, including two known genotypes D (n = 13) and EbpC (n = 5), and two novel genotypes named as CHN-F1 (n = 1) in a fox and CHN-R1 (n = 1) in a raccoon dog. Phylogenetic analysis revealed that all the four genotypes were the members of zoonotic group 1. Genotypes D and EbpC were found in humans previously. The findings of zoonotic genotypes of E. bieneusi in the foxes and raccoon dogs suggest these animals infected with E. bieneusi may pose a threat to human health.     

Diversity of Microsporidia,Cryptosporidium and Giardia in Mountain Gorillas (Gorilla beringei beringei) in Volcanoes National Park,Rwanda

Background

Infectious diseases represent the greatest threats to endangered species, and transmission from humans to wildlife under increased anthropogenic pressure has been always stated as a major risk of habituation.

Aims

To evaluate the impact of close contact with humans on the occurrence of potentially zoonotic protists in great apes, one hundred mountain gorillas (Gorilla beringei beringei) from seven groups habituated either for tourism or for research in Volcanoes National Park, Rwanda were screened for the presence of microsporidia, Cryptosporidium spp. and Giardia spp. using molecular diagnostics.

Results

The most frequently detected parasites were Enterocytozoon bieneusi found in 18 samples (including genotype EbpA, D, C, gorilla 2 and five novel genotypes gorilla 4–8) and Encephalitozoon cuniculi with genotype II being more prevalent (10 cases) compared to genotype I (1 case). Cryptosporidium muris (2 cases) and C. meleagridis (2 cases) were documented in great apes for the first time. Cryptosporidium sp. infections were identified only in research groups and occurrence of E. cuniculi in research groups was significantly higher in comparison to tourist groups. No difference in prevalence of E. bieneusi was observed between research and tourist groups.

Conclusion

Although our data showed the presence and diversity of important opportunistic protists in Volcanoes gorillas, the source and the routes of the circulation remain unknown. Repeated individual sampling, broad sampling of other hosts sharing the habitat with gorillas and quantification of studied protists would be necessary to acquire more complex data.     

Quantitative Evaluation of the Impact of Bather Density on Levels of Human-Virulent Microsporidian Spores in Recreational Water

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively. Enterocytozoon bieneusi spores were detected in 59% of weekend (n = 27) and 30% of weekday (n = 33) samples, and Encephalitozoon intestinalis spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples; P values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with P values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with Enterocytozoon bieneusi spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.