Case series of intestinal microsporidiosis in non-HIV patients caused by Encephalitozoon hellem

ABSTRACT

Intestinal microsporidiosis is most often caused by Enterocytozoon bieneusi, and to a lesser extent by species of the genus Encephalitozoon. Until now, Encephalitozoon hellem was not clearly known to induce disease restricted to the intestine, or rarely in HIV subjects or in tropical countries. We report here 11 cases of delineated intestinal microsporidioses due to E. hellem diagnosed in France in non-HIV patients. Briefly, all patients were immunocompromised. They all suffered from diarrhoea, associated in nearly 50% of cases with weight loss. Concerning treatment, 5/11 patients had a discontinuation or a decrease of their immunosuppressive therapy, and 4/11 received albendazole. All patients recovered. Five different genotypes were identified based on the rRNA ITS sequence.

Introduction

Microsporidia are obligate intracellular eukaryotes infecting a wide range of animals, including humans [1]. Human intestinal microsporidiosis mainly involves Enterocytozoon bieneusi, and less frequently microsporidia belonging to the Encephalitozoon genus. Among the latter, E. intestinalis is considered to be the most frequently responsible for intestinal infection, whereas E. hellem and E. cuniculi infections have been reported in extra-intestinal infections [1].

Here, we report 11 cases of delineated intestinal microsporidiosis caused by E. hellem collected as part of the French National Reference Center (NRC) for microsporidiosis since 2018.

Materials and methods

Ethics

This study was carried out under the supervision of the NRC for “Cryptosporidiosis, Microsporidia and other digestive protozoa” and benefited from agreements with the French Data Protection Agency (CNIL) and certification by the ethics committee for the use of the NRC database.

Cases identification and diagnosis confirmation

Forty-five French medical laboratories collaborate to the NRC for “Cryptosporidiosis, Microsporidia and other digestive protozoa,” including 35 university hospitals, 3 hospitals and 7 private medical laboratories. Microsporidiosis cases diagnosed in each of these centres are reported to the NRC, and the clinical and epidemiological data are compiled. The 11 cases included in the study were diagnosed by real-time PCR in 7 of these French centers (in-house or commercial PCR depending on the center). Stools were then sent to the NRC where microscopic examination was performed on stools (Uvitex-2B fluorescent dye), and diagnosis was confirmed by sequencing a part of the internal transcribed spacer (ITS) rDNA region, as previously described [2]. All sequences were deposited in GenBank (accession numbers: OQ750536 to OQ750546).

Results

The main characteristics of the 11 cases are reported in Table 1. The majority of cases were diagnosed in adult males (n = 8/11). All cases occurred in immunocompromised patients, mainly solid organ transplant (SOT) patients (n = 9/11). The two non-SOT cases were diagnosed respectively in a patient suffering from a primary immune deficiency and a patient with acute lymphoblastic leukemia. All cases presented with diarrhea sometimes associated with general digestive symptoms and weight lost. The symptoms of the majority of patients (n = 10/11) were limited to digestive tract. Only one patient (case #11) reported respiratory symptoms but he was simultaneously infected with influenza A and SARS-CoV2 viruses. Digestive symptoms lasted an average of 2 weeks [5 days to 4 weeks] before diagnosis. The majority of patients were hospitalized for further care (n = 8/10; 1 missing data). For SOT patients, treatment consisted of dose reduction or discontinuation of immunosuppressive therapy (n = 5/10), and/or administration of albendazole (n = 4/10). Three patients recovered without any specific care. All those for whom data were available (1 missing data) were cured.

Table 1.

Characteristics of the 11 patients of this report who presented delineated intestinal infection caused by Encephalitozoon hellem.

Case #	Year of diag-nosis	Sex, Age (yrs)	History	IS treatment	Symptoms	Symptoms duration at diagnosis	Diagnostic confirmation by NRC*: Ct value / nb spores per field	Other pathogens	Cares and treatment	Outcome at 3 months
#1	2018	F, 74	Kidney transplant	MMF	Diarrhea, abdominal pain, dehydration, weight loss, vomiting, nausea, fever	6 days	25 / 4	None	Hospitalization Discontinuation of IS treatment	Recovery
#2	2019	M, 47	Kidney transplant	MMF	Diarrhea, vomiting	5 days	26 / 4	None	Hospitalization Albendazole Decrease of IS treatment	Recovery
#3	2019	F, 62	Kidney transplant	MMF	Diarrhea, weight loss	1 month	34 / <1	None	Hospitalization Decrease of IS treatment	Recovery
#4	2019	M, 52	Primary immune deficiency	Corticosteroid	Diarrhea, nausea	1 month	34 / <1	Clostridioides difficile	Hospitalization	Recovery
#5	2020	M, 78	Kidney transplant	Ciclosporine	Diarrhea, weight loss, nausea	1 week	25 / 2	None	Hospitalization Albendazole Decrease of IS treatment	Recovery
#6	2021	M, 52	Liver transplant	n.a.	Diarrhea	n.a.	32 / 1	n.a.	n.a.	n.a.
#7	2021	M, 19	Leukemia (ALL B relapse)	Corticosteroid Methotrexate Chemo-therapy (VANDA)	Diarrhea	1 week	20 / 10	None	Hospitalization	Recovery
#8	2021	M, 35	Kidney transplant	Corticosteroid Tacrolimus MMF	Diarrhea, weight loss	1 month	34 / <1	Cryptosporidium	Hospitalization Albendazole Rifaximine	Recovery
#9	2022	M, 33	Kidney transplant	Tacrolimus MMF	Diarrhea	3 weeks	22 / 1	None	Decrease of IS treatment	Recovery
#10	2022	M, 58	Heart transplant	Tacrolimus MMF	Diarrhea	1 week	22 / 5	None	Albendazole	Recovery
#11	2022	F, 76	Kidney transplant	Tacrolimus MMF	Diarrhea, fever, weight loss, respiratory symptoms	2 weeks	25 / 1	Influenza A and SARS-CoV2 infections	Hospitalization	Recovery

Notes: ALL: acute lymphoblastic leukemia; F: female; HSCT: hematopoietic stem cell transplantation; IS: immunosuppressive; M: male; MMF: mycophenolate mofetil; n.a.: not available; nb: number; NRC: National Reference Center for microsporidiosis; VANDA regimen: etoposide, asparaginase, mitoxantrone, dexamethasone; yrs: years; *: cycle threshold (Ct) value for qPCR and number (nb) of spores per field of a fecal smear with magnification ×1000 for Uvitex-2B.

The source of contamination could not be identified for any of the cases. Two patients had recently travelled to the tropics before reporting symptoms (case #7 lived in Guyana and case #8 returned from a trip to Mali).

According to ITS genotypes defined by Xiao et al. [3], E. hellem isolates from cases 4, 6, 8 and 11 belong to genotype 1 (Figure 1, A); from case 2 to genotype 2B; from cases 1, 3 and 5 to genotype 2C. Isolates from cases #7 and 10 were identical to a strain previously reported in Hummingbirds (AF272836) and closely related to genotype 1. Isolate from case #9 was identical to the PV7/95 (AY171241) strain isolated from an HIV patient, and related to genotype 2.

Figure 1.

Phylogenetic analysis of ITS sequences of Encepahlitozoon hellem isolates from the present study and reference sequences (outgroup: Encephalitozoon cuniculi). The evolutionary history was inferred by using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model with a discrete Gamma distribution (10,000 replicates). Best model determination and phylogenetic analyses were performed using MEGA 7 software [4].

Discussion

E. hellem is essentially encountered in birds, even though it was also detected in mammalian [3]. In humans, E. hellem was rarely reported to be responsible for intestinal microsporidiosis (cases reviewed in Table 2). These previous cases involved mainly HIV/AIDS patients and/or those who had visited countries with a low sanitary level [5–13]. Furthermore, few clinical data are available in previous studies, making it impossible to conclude whether E. hellem infection is restricted to the intestinal tract.

Table 2.

Literature review of cases where E. hellem was detected in stool.

Article type	Year (if known)	Context	Symptoms	Diagnostic tools	Country	Nb of positive patients for E. hellem [nb of positive patients for microsporidia]	Refs.
Cohort study	1998	Diarrhea on return from different tropical areas	Diarrhea	Uvitex 2B staining and PCR	Singapore for the 2 E. hellem	2/148 (co-infected with E. bieneusi) [9/148]	[10]
Cohort study	n.a.	HIV/AIDS	Chronic diarrhea	Oligonucleotide microarray	USA	2/20 (co-infected with E. cuniculi or E. cuniculi + E. intestinalis) [12/20]	[12]
Cohort study	between 1992 and 2003	HIV/AIDS	Diarrhea	FISH	USA	2/110	[11]
Cohort study	n.a.	Breast cancer patients, 6 rounds of treatment	Gastritis	Modified trichrome stain and nested PCR for positive specimen	Malaysia	1/311 (co-infected with E. intestinalis) [68/311]	[9]
Cohort study	n.a.	HIV/AIDS or myeloma	n.a.	Modified trichrome stain and PCR	Tunisia	1/86 [9/86]	[13]
Cohort study	between 2006 and 2009	HIV/AIDS (most of patients had not received cART)	Chronic or acute diarrhea	Histochemical staining or PCR and sequencing	Russia	1/159 [30/159]	[7]
Case report	2006	CD4+ T-cell prolymphocytic leukemia, 12 courses of alemtuzumab and pentostatin	Burning urination, diarrhea, fever, general weakness, anorexia, weight loss	Uvitex 2B and Weber’s modified trichrome stains: spores in urine and stools; then SSU rRNA sequencing	France	/	[6]
Cohort study	n.a.	Immunocompromised patients (AIDS, Hodgkin lymphoma, hemodialysis for the 4 E. hellem cases)	n.a.	Modified trichrome stain and multiplex/nested PCR + sequencing	Iran	4/310 [88/310]	[14]
Cohort study	n.a.	Leukemic children on chemotherapy	n.a.	Modified trichrome stain and PCR + sequencing	Egypt	3/100 [29/100]	[8]

Notes: Articles are listed by year of publication.

F: female; FISH: fluorescence in situ hybridization; IS: immunosuppressive; M: male; n.a.: not available; nb: number; Ref: reference; SSU: small subunit

We describe here for the first time a cases series of delineated intestinal infections caused by E. hellem. In our study, these infections only occurred in immunocompromised patients but no patient was HIV-seropositive. The overrepresentation of SOT patients reflects the immunocompromised population currently at risk for intestinal microsporidiosis in France. We recognize that it would have been necessary to search for E. hellem in other specimens than stool to assess intestinal delineated infection, but none of patients presented extra intestinal symptoms justifying urinary or respiratory samplings, by example. One patient (case #8) benefited from an extension assessment with microsporidia search in two different urine samples which were negative. Interestingly, all samples positive for E. hellem which have been referred to the NRC for microsporidiosis since 2018 were these 11 cases. So, extra intestinal infections that were reported in the literature can probably be considered as extremely rare events, and/or, that non-HIV immunocompromised subjects are less frequently at risk of extra-intestinal infections. Moreover, as diagnosis of the 11 cases reported here were performed in different laboratories, the PCR techniques were not the same and some have identified the presence of E. intestinalis. Indeed, we have previously demonstrated that E. hellem is misidentified as E. intestinalis with some current commercial or in-house PCRs assay [14]. So we hypothesized that the number of E. hellem intestinal infection cases has been underestimated until now. Since 2018, the French NRC for microsporidiosis recorded 12 cases of E. intestinalis infections (confirmed by ITS sequencing), making E. hellem as frequent as E. intestinalis for Encephalitozoon-associated intestinal microsporidioses.

We also explored the hypothesis of particular E. hellem genotypes that could be involved in delineated intestinal infections. Indeed, several E. hellem genotypes have been previously described based on the rRNA ITS sequence [15, 16]. However, among the 11 new cases we reported, we identified five different ITS genotypes, some of them being also reported from extra-intestinal infections in the literature [17].

In summary, this study highlights that E. hellem may be a cause as frequent as E. intestinalis for delineated intestinal microsporidioses in immunocompromised patients in France.

Funding Statement

This work was supported in part by the “Santé Publique France” and by the “Direction de la Recherche Clinique et de l’Innovation” of the University Hospital of Clermont-Ferrand (France).

Disclosure statement

No potential conflict of interest was reported by the author(s).