Microsporidia cause opportunistic infections in highly immunodeﬁcient individuals. Few studies on the epidemiology of these infections have been conducted in France. Between 2014 and 2016, we undertook a study to estimate the prevalence and circulating genotypes of this fungus-related micro-organism among the population of Strasbourg University Hospital. Samples were collected from hospitalized patients and analyzed using microscopy and molecular assays. Strains from positive subjects were sequenced for genotyping. Only 7/661 patients (1.1%) were positive for microsporidia, and the only species identiﬁed was Enterocytozoon bieneusi. Two patients presented immunodeﬁciency linked to AIDS, and ﬁve transplant recipients presented immunodeﬁciency linked to immunosuppressive therapies. Only ﬁve patients received speciﬁc antimicrosporidial treatment, but clinical outcomes were good in all cases. We identiﬁed four genotypes: A and D in patients with AIDS, and C and S9 in transplant recipients. To date, genotype S9 has been described only once. This genotype is similar to those found in farm animals in China. Because some of these animals have been introduced to Central Europe, we postulate that this genotype might be of Asian origin. Thus, genotyping microsporidial strains may be of epidemiological and clinical interest to identify potential outbreak sources depending on the infecting strains. Introduction initially placed Nosema bombycis in the fungal group, Microsporidia are a group of unicellular fungi living as Schizomycetes, microsporidia were considered protozoans 1 1 obligate intracellular parasites . The ﬁrst microsporidial for more than a century . Recently, phylogenetic analyses species to be described, Nosema bombycis, was identiﬁed of ﬁve gene sequences (mitochondrial HSP70, TATA-box by Carl Wilhelm von Nägeli in 1857 as the causative protein, RNA polymerase II, and α-tubulin and β-tubulin) agent of a silkworm disease. Since then, approximately have supported classifying microsporidia within the fungi 1 1 1000–1500 microsporidial species have been described . or as a sister-group with a common ancestor . Over the past 150 years, microsporidia classiﬁcation has Until the 1970s, microsporidial species were rarely been widely discussed, based on morphological, bio- recognized as causes of human pathology . With the HIV chemical, and genetic characteristics. Although Nägeli epidemic emerging in the ﬁrst half of the 1980s, micro- sporidia, which cause opportunistic infections in highly immunodeﬁcient individuals, became more frequent and visible, concomitant with physicians’ increasing knowl- Correspondence: Valentin Greigert (email@example.com) 1 1 Infectious Diseases Department, Hôpitaux Civils de Colmar, 39 Avenue de la edge of this condition . Thus, diverse symptoms were Liberté, Colmar, France associated with microsporidial infections, most commonly Institute of Parasitology and Tropical Diseases, EA 7292, University of digestive disorders, but many other unusual symptoms as Strasbourg, 3 rue Koeberlé, Strasbourg, France Full list of author information is available at the end of the article. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1234567890():,; 1234567890():,; Greigert et al. Emerging Microbes & Infections (2018) 7:97 Page 2 of 7 well . Four species primarily cause human infections: Table 1 Underlying conditions of patients tested for intestinal microsporidiosis Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi. Underlying immunodeﬁciency 613 (92.7%) Few studies on the epidemiology of these infections have been conducted in France, and most of these studies Solid organ transplant 404 (61.1%) were on microsporidial infections in HIV-infected Solid cancer 49 (7.4%) patients. However, current patients undergoing immu- Malignant hemopathy 39 (5.9%) nosuppressive therapies, such as transplant recipients, account for many of the immunocompromised patients, HIV infection 39 (5.9%) and microsporidial infections have been described under 3 T4 cells <200/mm 15 (2.3%) these conditions . In this retrospective study, we eval- T4 cells >200/mm 24 (3.6%) uated the frequency of these infections in Eastern France Hematopoietic stem cell transplant 33 (5.0%) by analyzing cases diagnosed between January 2014 and December 2016 at the Strasbourg University Hospital. We Autoinﬂammatory or autoimmune disease 29 (4.4%) were interested in epidemiological, clinical, and micro- Kidney failure 5 (0.8%) biological features, including genotyping, to determine the Corticosteroid treatment 4 (0.6%) current burden and characteristics of these infections in Other infection 4 (0.6%) Eastern France. Malnutrition 3 (0.4%) Results Humoral immune deﬁciency 3 (0.4%) Clinical features Alcoholic liver cirrhosis 1 (0.2%) Between January 2014 and December 2016, 661 samples No immunodeﬁciency 42 (6.4%) were sent to the laboratory of the University Hospital of Strasbourg for microsporidia testing. Of these, 613 No information 7 (1.1%) (92.7%) came from immunocompromised patients, Total 662 including most organ transplant recipients (61.1%) The total is 662 because one subject infected with HIV also received a (Table 1). Samples were taken from 281 females (42.5%) hematopoietic stem cell transplant and 380 males, with an average age of 52.27 years (range 16 days–92 years), and 57 samples (8.6%) were from patients under 18-years old. Only seven patients (1.1%) Five patients received speciﬁc antimicrosporidial treat- were positive for microsporidia, and the only species ment with four regimens (Table 2). We could not access detected was E. bieneusi. The positive individuals’ ages treatment information for one patient because of ranged from 7 to 63 years, and there were 5 males and 2 incomplete medical records, and two patients received no females (Table 2). speciﬁc treatment for the E. bieneusi infection; however, All patients were immunodeﬁcient; two patients were these three subjects were treated for other infections: one linked to HIV infection with AIDS (C3 stage), and the with azithromycin, one with azithromycin and trimetho- other ﬁve were linked to immunosuppressive therapies. prim-sulfamethoxazole, and the last with spiramycin, The two patients with HIV had very low CD4 counts (38 cefotaxime, vancomycin, and valganciclovir. and 61/mm ). For patients undergoing immunosuppres- The clinical outcomes were good with digestive symp- sive therapies, these treatments were administered to toms being resolved in all cases. Three patients received a prevent organ rejection after transplantation (four kid- second parasitological control examination 2–6 weeks neys, one heart), and all ﬁve patients were receiving at after diagnosis. Two remained positive at this least a 2-drug regimen, including mycophenolate, tacro- examination. limus, everolimus, ciclosporin, and prednisone (Table 2). All seven patients presented digestive symptoms with Microbiological features acute diarrhea (three patients), chronic diarrhea (three Microsporidia was diagnosed using two polymerase patients), or “yellow” stool (one patient). Six patients chain reactions (PCRs), the ﬁrst to detect microsporidial presented with biological inﬂammatory syndrome with species from the Enterocytozoon and Encephalitozoon moderately elevated C-reactive protein (CRP) ranging genera and the second speciﬁc for E. bieneusi only from 24.5 mg/l to 109 mg/l. All positive patients pre- (Fig. 1). All subjects positive for microsporidia were sented comorbidities (tuberculosis, hepatitis B and C, infected with E. bieneusi. Of the 661 samples examined, pneumonia, pericarditis, pneumocystosis, macrophage 46 (6.9%) were positive for other parasites. Only one activation syndrome, cryptococcosis, and transplant (subject 5) was coinfected with E. bieneusi, Blastocystis rejection). spp., and Chilomastix mesnili. Eight patients were Greigert et al. Emerging Microbes & Infections (2018) 7:97 Page 3 of 7 coinfected with 2–3 parasites but not E. bieneusi. All other patients positive for parasites were mono-infected (detailed in Table 3). To genotype the isolated strains, another PCR was performed using primers targeting internal transcribed spacers (ITS) between small-subunit rRNA and large- subunit rRNA genes . This procedure allowed compar- ison with published sequences in the GenBank database. All sequences were genetically similar, but four genotypes were identiﬁed (Fig. 2). One HIV-positive patient was infected with E. bieneusi genotype D, the other with genotype A. Among the ﬁve patients receiving immuno- suppressive therapies, three were infected with E. bieneusi genotype C (all were kidney-transplant recipients) and two with genotype S9 (one kidney transplant and one heart transplant recipient). Subject 2 tested positive twice for genotype C. Subject 5 tested positive twice for E. bieneusi, with genotype A for the ﬁrst sample; however, the volume of the second sample was insufﬁcient and could not be genotyped. Discussion We report seven cases of intestinal microsporidiosis due to E. bieneusi in immunodeﬁcient patients. Most patients were transplant recipients with marked immunodeﬁ- ciency mediated by anti-organ-rejection therapies. With the development of HIV protease inhibitors in 1995 and the use of combined antiretroviral therapy (cART) , subjects with HIV have become less susceptible to 8–12 microsporidial disease . More recently, powerful immunosuppressive therapeutics were used to prevent organ rejection after transplantation , putting transplant recipients at risk of infection . In addition, HIV-positive patients with very low CD4+ cell counts remain at risk. All patients who were screened for microsporidia pre- sented symptoms attributable to E. bieneusi infection, which is known to cause a relatively localized disease, with a tropism for the gastrointestinal tract . Diagnoses were performed using microscopy (until 2015) and PCR; however, this search was positive only 10 times for 7 patients over 3 years, with 661 tests performed. Thus, microsporidial disease seems to be an uncommon diag- nosis at our hospital. Only four of the seven diagnosed patients were speci- ﬁcally treated for E. bieneusi infection, with four drug regimens using nitazoxanide and/or albendazole. Two patients remained untreated and we found no information on the last patient. Outcomes were good in all cases. Currently, France has no ofﬁcial treatment recommen- dation for intestinal microsporidiosis or E. bieneusi infection; the most commonly used drugs are nitazox- 15, 16 17, 18 anide and albendazole . However, several suc- cessful treatments have been reported using fumagillin for intestinal microsporidiosis due to E. bieneusi in transplant Table 2 Patients positive for E. bieneusi Subject Age Sex Immuno-deﬁciency Symptoms Genotype Diagnostic method Speciﬁc treatment Clinical Biological outcome outcome 1 28 F AIDS Acute diarrhea D PCR microscopy ALBENDAZOLE 400 mg b.i.d., 3 days Success No control 2 55 M Immune-suppressive Acute diarrhea C PCR NITAZOXANIDE 500 mg b.i.d., 14 days + ALBENDAZOLE Success Failure therapy 400 mg b.i.d., 10 days 3 58 F Immune-suppressive Chronic diarrhea S9 PCR None Success No control therapy 4 7 M Immune-suppressive Chronic diarrhea S9 PCR Unknown Success No control therapy 5 43 M AIDS “Yellow” stool A PCR ALBENDAZOLE 400 mg b.i.d., 21 days Success Failure 6 57 M Immune-suppressive Acute diarrhea C PCR NITAZOXANIDE 500 mg b.i.d., 15 days Success No control therapy 7 63 M Immune-suppressive Acute diarrhea C PCR None Success Success therapy M male, F female, AIDS acquired immune deﬁciency syndrome Clinical outcomes: “success” refers to the resolution of all digestive symptoms. Biological outcome: “success” and “failure” refer to the absence or presence, respectively, of microsporidia species from the Enterocytozoon or Encephalitozoon genera in patient stool samples Greigert et al. Emerging Microbes & Infections (2018) 7:97 Page 4 of 7 Table 3 All parasite results from 661 patient stool samples Number (/661) Prevalence (%) Parasites 46 6.9 Protozoa 37 5.6 Blastocystis spp. 17 2.6 Amebas 7 1.1 Entamoeba coli 1 0.15 Entamoeba dispar 2 0.3 Fig. 1 Diagnostic procedure for intestinal microsporidiosis Entamoeba hartmanni 1 0.15 Endolimax nana 2 0.3 19–23 recipients . Furthermore, in most previously reported Undeﬁned ameba 1 0.15 cases, immunosuppressive therapies were tapered or dis- Flagellates 7 1.1 continued at the same time treatment was introduced . Dientamoeba fragilis 1 0.15 Similarly, in our study, cART was initiated in HIV- infected patients, and immunosuppressive therapies were Chilomastix mesnili 2 0.3 tapered in transplant recipients. Since introducing cART, Giardia intestinalis 4 0.6 many positive outcomes have been reported for intestinal Cystoisospora belli 1 0.15 microsporidiosis after immune restoration, with no other 8–12 Cryptosporidium spp. 12 1.8 speciﬁc treatment . However, a recent publication showed that intestinal microsporidiosis due to E. bieneusi Helminths 4 0.6 in a stem cell transplant recipient was successfully treated Nematodes 3 0.45 with fumagillin and avoided modiﬁcations of immuno- Ancylostomidae 1 0.15 suppression . In any case, patient immunity seems to be Enterobius vermicularis 1 0.15 a key element in curing this infection. Finally, given the low prevalence of E. bieneusi infections in patients with Trichuris trichiura 1 0.15 diarrhea in our study, directly implicating this micro- Tenia 1 0.15 organism in intestinal symptom development in these Diphyllobothrium latum 1 0.15 patients remains uncertain. All samples analyzed were Fungi 7 1.1 from patients experiencing diarrhea, but only 7 (1.1%) were positive for E. bieneusi. These patients may have Enterocytozoon bieneusi 7 1.1 only been carriers of this microorganism. These elements raise the question of the value of diagnosing intestinal microsporidiosis and using speciﬁc treatments. Netherlands . This genotype is very similar to two other We identiﬁed E. bieneusi strains that were genetically recently described genotypes, NCR1 and NCF2, found in similar to four described genotypes: A, C, D, and S9. The raccoon dogs (Nyctereutes procyonoides) farmed in most predominant genotype was C, found in three Northern China . Other highly similar strains to these patients, all of whom were kidney transplant recipients. three genotypes have mostly been found in animals in 29–31 Genotype C is the most common genotype in kidney China and recently in wild animals, especially wild transplant patients, with a high speciﬁcity for this immune boars (Sus scrofa) and raccoons (Procyon lotor) introduced 5, 24 25 32, 33 background , as well as for liver transplant recipients . in Central Europe . With Strasbourg being on the We described one patient infected with genotype A and German border, these strains are likely to circulate in our one with genotype D, both being HIV-infected with AIDS. region, possibly after having been introduced from Asia. It These genotypes are frequently observed in HIV-infected would be interesting to genotype more strains isolated patients ; however, we did not isolate the most commonly from patients from the Rhine valley and Central Europe to 5, 26, 27 described genotype in this HIV setting, genotype B . assess whether these strains occur frequently in those The fourth genotype was S9, carried by two patients areas. undergoing immunosuppressive therapies, one being a Furthermore, outbreaks related to environmental con- heart transplant recipient, the other a kidney transplant tamination can occur in immunodeﬁcient and immuno- recipient. To our knowledge, this genotype has only been competent individuals . Although infections are believed 1, 35 described once, in a patient with ulcerative colitis in the to occur via ingestion , E. bieneusi can infect other Greigert et al. Emerging Microbes & Infections (2018) 7:97 Page 5 of 7 Fig. 2 Bayesian inference phylogeny of small-subunit rRNA gene internal transcribed spacer from the strains isolated from patients and similar strains estimated using the program MrBayes with the general time reversible (GTR) substitution model. Numbers under the lines show relative distances between the strains. Study patients are marked 1–7. Reference sequences were those used by Santín et al. for E. bieneusi nomenclature consensus and are marked with their GenBank accession number mammals, birds, insects, and arthropods, making the context, genotyping strains isolated from patients may contamination source difﬁcult to ascertain, with a help identify environmental sources that could potentially potential for zoonotic and arthropod-borne transmis- initiate outbreaks. Finally, we identiﬁed strains similar to sion . Thus, this makes it difﬁcult to recommend pre- uncommon genotypes that may have been introduced ventive behaviors to patients potentially at risk of through animals imported from Asia. This would show microsporidial infection, and strain genotyping should be the importance of human activities in spreading human routinely performed to identify environmental sources and animal pathogens. that may cause outbreaks. In conclusion, given its epidemiological characteristics Materials and methods linked to immunocompromised hosts, intestinal micro- Sample collection sporidiosis due to E. bieneusi is an uncommon but All samples were collected from patients at the Uni- potentially emerging disease with increased use of versity Hospital of Strasbourg per standard procedures immunosuppressive treatments. Our study shows that, in over a 3-year period (January 2014 to December 2016). our region, transplant recipients are the main population Subjects were included when their clinician speciﬁcally at risk of infection. The absence of treatment recom- requested testing for microsporidia. mendations leaves physicians with the choice of a drug regimen that does not appear to affect the usually favor- Microscopic examination able course of infection, and patient immunity might be a A ﬁrst microscopic examination was performed without key element in triggering and curing this infection. In this staining shortly after stool collection. Samples were then Greigert et al. Emerging Microbes & Infections (2018) 7:97 Page 6 of 7 examined after merthiolate-iodine-formaldehyde staining Conﬂict of interest The authors declare that they have no conﬂict of interest. and concentration (Faust-Ingalls method). From January to December 2014, all samples were also stained with Weber’s modiﬁed trichrome for microsporidial spore Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in staining before microscopic examination by a trained published maps and institutional afﬁliations. parasitologist. This step was discontinued in 2015 due to a change in legislation prohibiting use of the toxic reagents Received: 5 February 2018 Revised: 16 April 2018 Accepted: 29 April 2018 necessary for Weber’s staining and because no superiority in sensitivity or speciﬁcity has been found compared with PCR . References Molecular assays 1. Franzen, C. Microsporidia: a review of 150 years of research. Open Parasitol. J. 2, An aliquot of each stool sample was stored at −20 °C 1–34 (2008). until molecular diagnosis. DNA was extracted using the 2. Nageli, K. W. Uber die neue Krankheit der Seidenraupe und verwandte Organismen. Bot. Z 15,760–761 (1857). 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