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Journal of Clinical Microbiology, July 1998, p. 1882-1885, Vol. 36, No. 7
Laboratory of Microbiology,
Received 20 January 1998/Returned for modification 20 February
1998/Accepted 26 March 1998
To determine the types of Enterocytozoon bieneusi
strains associated with intestinal microsporidiosis, we developed a
rapid and efficient approach for typing parasites obtained from stool specimens by PCR-restriction fragment length polymorphism (PCR-RFLP). Typing was based on DNA polymorphism of the ribosomal DNA internal transcribed spacer (ITS) region of E. bieneusi. RFLPs
generated with two restriction enzymes (NlaIII and
Fnu4HI) in PCR-amplified ITS products were used to classify
strains into different lineages. This approach was successfully used to
differentiate 78 strains that had been obtained from the stools of 65 patients with intestinal microsporidiosis. Among the 78 strains, we
found four genetically unrelated lineages, showing the genetic
diversity of E. bieneusi. Type I strains of E. bieneusi were found in a majority of the samples, accounting for
51 (78%) of the 65 microsporidiosis cases. In contrast, type II, III,
and IV strains were found in only 8 (12%), 3 (5%), and 3 (5%) cases,
respectively. All strains of E. bieneusi we have tested so
far fall into one of four different lineages, and this study shows that
human intestinal microsporidiosis is most often associated with type I
strains. PCR-RFLP analysis of the ITS region of E. bieneusi
should be useful for epidemiological studies.
Microsporidia are obligate
intracellular protozoan parasites that can infect vertebrates as well
as invertebrates. Over 1,000 species of microsporidia have been
described, but reports of human infections were rare before the AIDS
epidemic. Since then, microsporidia have been recognized as
opportunistic pathogens in patients with AIDS. Different
species of microsporidia have been shown to infect humans, and among these species, Enterocytozoon
bieneusi (7) is by far the most frequently identified.
E. bieneusi infection is responsible for chronic
diarrhea and wasting in immunocompromised patients, such as
patients with AIDS and organ transplant recipients (2, 15, 17,
23). Substantial variation in the progression and severity of
disease is observed among cases of intestinal microsporidiosis, and these differences are presumably
due to several factors, including host immune defenses and phenotypic differences in parasite strains (23).
However, modes of transmission and sources of human infection by
E. bieneusi are largely unknown (11, 23).
Epidemiologic investigations of a number of infectious diseases have
shown that amplification methods could be extremely valuable tools for
laboratory-based investigations (20). This approach has
proven successful in defining different strains of
Encephalitozoon cuniculi, a zoonotic microsporidian that can infect humans and a wide
variety of other mammals (6, 8, 10, 14). At least three
strains of this species have been defined, on the basis of the number
of 5'-GTTTT-3' repeats present in the internal transcribed spacer
(ITS) region of the ribosomal DNA. We wished to study the genetic
diversity of E. bieneusi strains by a similar approach. In
this report, we describe the development of an amplification-based
assay for genetic analysis of the ITS region of E. bieneusi
that should be useful for epidemiological studies.
Patient specimens.
Seventy-eight stool specimens were
obtained over a 4-year peiod (1994 to 1997) from 65 patients with
intestinal microsporidiosis seen at the
Department of Infectious Diseases, Hôpital Saint-Louis, Paris,
and the Hôtel-Dieu, Lyon, France (15-17). All
patients were immunocompromised. Of these patients, 63 were infected
with human immunodeficiency virus (HIV) and 2 were not HIV infected but
had undergone heart-lung and kidney transplantation. The diagnosis of
microsporidial infection in these patients was made by
the detection of typical spores in stools by two different techniques as previously described (16). The species-level
identification of E. bieneusi was made by transmission
electron microscopy analysis of duodenal biopsy samples (38 patients)
and/or by PCR with DNA extracted from duodenal biopsy samples or stools
(44 patients) with primer sets specific for the small-subunit rRNA gene
of E. bieneusi as previously described (4, 12).
DNA extraction.
Fresh stools were inactivated for 2 h
at 65°C and stored at PCR amplification.
Primers for PCR were chosen to amplify
the ITS region of E. bieneusi. The forward primer Eb.gc
(5'-TCAGTTTTGGGTGTGGTATCGG-3'), complementary to positions 1 to 22, was designed by using the GenBank sequence of E. bieneusi (22, 24) (accession no. L20290). The reverse
primer Eb.gt (5'-GCTACCCATACACACATCATTC-3') was designed to
be complementary to positions 189 to 210 of the GenBank sequence of
E. bieneusi (22, 24) (accession no. L20290). The
PCR was performed in 50 µl of reaction mixtures that contained the following: 2.5 µg of each primer/ml, 200 µmol of each
deoxynucleoside triphosphate/liter, 75 mmol of Tris-HCl (pH 9.0)/liter,
20 mmol of (NH4)2SO4/liter, 0.01% Tween 20, 2 mmol of MgCl2/liter, and 2 U of heat-stable DNA polymerase
(Goldstar; Eurogentec, Seraing, Belgium). DNA amplification was
performed on a Hybaid (Teddington, Middlesex, United Kingdom) touch
down. The amplification procedure included 3 min of initial
denaturation at 94°C (1 cycle), followed by 30 s of denaturation
at 94°C, 30 s of annealing at 55°C, and 60 s of extension
at 72°C for 40 cycles. A 10-min extension at 72°C was used after
the 40 cycles. Each set of reactions included a negative control (a
tube containing all the reagents but not the template DNA), to ensure
the absence of contamination of samples during analysis, and a positive
control. Culture spores of Encephalitozoon intestinalis,
E. cuniculi, and Encephalitozoon hellem were also tested to assess the specificity of the primer set Eb.gc-Eb.gt. A
10-µl aliquot from each reaction mixture was run on a 1.2% agarose gel (agarose standard; Bioprobe, Montreuil, France) and was stained with ethidium bromide to visualize the amplified-PCR products under UV
illumination.
DNA sequencing of PCR products.
To assess genetic diversity
among stool isolates, amplification products of the ITS regions from
six independent samples were gel purified from 1.2% agarose gels run
in 1× Tris-borate-EDTA with a Geneclean II DNA purification kit (Bio
101, Vista, Calif.) by following the manufacturer's protocol. For each
specimen, the nucleotide sequence of the amplified product was
determined by a commercial company (Euro Sequence Genes Services, Evry,
France) on an ABI 377 sequencer using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.). Both
strands were sequenced with the primers used for the PCR. The
nucleotide sequence of each specimen was compared to the others and
with the E. bieneusi ITS GenBank sequence (accession no.
L20290) with Sequence Navigator software (Perkin-Elmer, Applied
Biosystems Division).
Restriction endonuclease digestion of PCR products.
Two
restriction endonucleases, NlaIII and Fnu4HI (New
England Biolabs, Beverly, Mass.), were selected from the DNA sequencing data with DNA Strider 1.2 software (Commisariat Energie Atomique, Gif-sur-Yvette, France) to digest PCR products obtained from
amplification of stool DNA extracts (Fig.
1). Ten microliters of PCR product was
digested in two separate tubes with 10 U of each restriction enzyme in
a final volume of 20 µl, as described previously
(18). The restriction digests were electrophoresed through
polyacrylamide gels (Gibco BRL, Paisley, Scotland), stained with
ethidium bromide, and photographed under UV illumination. All
restriction fragment length polymorphism (RFLP) patterns were compared
visually and classified as matching if the numbers and molecular
weights of the bands were identical.
PCR amplification of the E. bieneusi ITS
region.
DNA was easily extracted from all stool specimens. Use of
the Eb.gc-Eb.gt primer set resulted in efficient amplification of all
specimens tested. For three (5%) stool specimens, however, a 10-fold
dilution of released DNA was necessary to remove PCR inhibitors. No
other modification of the protocol was made for these three samples,
and the results of amplification were as unambiguous as those obtained
with the other samples. The positions of the primer set Eb.gc-Eb.gt
within the ITS sequence predicted that a 210-bp fragment would be
generated. Fragments of the appropriate size were visualized on agarose
gels following PCR of released DNA obtained from all stool specimens,
but not from culture spores of E. intestinalis,
E. cuniculi, or E. hellem (data not
shown).
Nucleotide sequence of the ITS regions of six E. bieneusi isolates.
The sequences of four amplified DNA
fragments obtained from stool samples matched the sequence of the
GenBank database (accession no. L20290) for the ITS region of
E. bieneusi, confirming that we successfully amplified
E. bieneusi from the stools of our patients. The
nucleotide sequences of the two other amplified DNA fragments, however,
showed one and seven mismatches (97% identity) when compared with the
E. bieneusi ITS GenBank sequence. The E. bieneusi ITS GenBank sequence was classified as type I, as were
the four amplified fragments that matched the ITS E. bieneusi GenBank sequence. The amplified fragment that showed one
mismatch was still classified as type I (no restriction site), and the
amplified fragment that showed seven mismatches was classified as type
II.
Typing analysis.
RFLP analysis of the amplification products
of the ITS region was then performed on the 78 E. bieneusi stool isolates. For all samples, digestion of amplicons
with NlaIII and Fnu4HI produced distinctive
fragments detectable in ethidium bromide-stained polyacrylamide gels
(Fig. 2). The sizes of the DNA fragments
in the various RFLP profiles were all in accordance with the positions
of the restriction enzyme cleavage sites on the ITS region as deduced
from their sequences (Table 1). Among the
78 stool specimens we found four genetically unrelated lineages. Type I
strains of E. bieneusi were found in a majority of the
samples, accounting for 51 (78%) of the 65 microsporidiosis cases (Table
2). In contrast, type II, III, and IV
strains were found in only 8 (12%), 3 (5%), and 3 (5%) of the 65 cases, respectively (Table 2). No association was seen between the
types of the parasite strains and clinical presentation, and no mixed
infection was noted. However, stool specimens from 2 non-HIV-infected
patients with intestinal microsporidiosis contained a
type II strain, while only 6 of 63 (9.5%) HIV-infected patients were
found to shed type II strains in their stools (Table 2).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Determination of Types of Enterocytozoon bieneusi
Strains Isolated from Patients with Intestinal
Microsporidiosis
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
20°C until DNA extraction. Microsporidian
DNA was extracted from 100 µl of filtrated stool suspension by the
guanidium thiocyanate-based method described by Boom et al.
(1), as adapted for stool samples by van der Hoek et al.
(21). A 100-bp DNA size marker was used as a control for DNA
extraction.
View larger version (18K):
[in a new window]
FIG. 1.
Diagram of the E. bieneusi ITS region
showing the locations of the primers used for PCR amplification of the
210-bp fragment and the polymorphic restriction sites used for
identification of types.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (53K):
[in a new window]
FIG. 2.
RFLP analysis of PCR products of strains of the four
E. bieneusi lineages generated with primer set
Eb.gc-Eb.gt and digested by either NlaIII or
Fnu4HI. (A) Lanes: M, molecular size markers corresponding
to pBR322 DNA digested by MspI (New England Biolabs); 1 to
3, PCR products from type I strains without digestion and following
digestion by NlaIII and Fnu4HI, respectively; 4 to 6, PCR products from type II strains without digestion and following
digestion by NlaIII and Fnu4HI, respectively. (B)
Lanes: M, molecular size markers; 1 to 3, PCR products from type III
strains without digestion and following digestion by NlaIII
and Fnu4HI, respectively; 4 to 6, PCR products from type IV
strains without digestion and following digestion by NlaIII
and Fnu4HI, respectively. The different strains can be
distinguished by comparing the numbers and sizes of bands.
TABLE 1.
Sizes of restriction enzyme digests
TABLE 2.
Prevalence of E. bieneusi strain types in
stool specimens
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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In the present report, we describe a rapid and efficient method for determining the types of unrelated human E. bieneusi strains isolated from stool specimens of patients with intestinal microsporidiosis.
Different PCR assays have been used previously for detection of E. bieneusi in clinical samples from patients with intestinal microsporidiosis, using the single-stranded rRNA gene as the target (3, 4, 9, 12). This locus, however, is not sufficiently polymorphic to allow strain typing (9, 22, 24), and E. bieneusi was thought therefore to be a relatively homogeneous entity. Consequently, we chose to develop a PCR-RFLP assay based on the putative polymorphism of the ITS region of E. bieneusi to study the genetic diversity of E. bieneusi. This approach has proven successful for the typing of E. cuniculi strains (6, 8, 10, 14).
In our study, the presence of restriction sites in the ITS region of E. bieneusi permitted the identification of individual strains by DNA fingerprinting with RFLP analysis. Indeed, PCR amplification of the ITS region of E. bieneusi, followed by RFLP analysis, allowed us to assign all 78 isolates tested to one of four different lineages. Also, the same strains were found in sequential stool specimens from the same patients, suggesting the clonal nature of E. bieneusi infection in the gastrointestinal tract. Among the 65 patients we studied, type I strains of E. bieneusi were much more prevalent than type II, III, or IV strains. It is, however, not clear yet whether this high prevalence of type I strains in humans simply reflects a common source of strains that lead to human infection. Alternatively, this high prevalence could be related to the pathogenicity of this strain in a particular type of host, namely, HIV-infected patients. Indeed, stool specimens of the two immunocompromised patients without HIV infections contained only type II strains, and this type occurred in only 9% of HIV-infected patients (Table 2). However, no evident association between clinical symptoms and strain type was seen in our study, as all of our patients were symptomatic.
Interestingly, E. bieneusi has recently been found in stool specimens from a pig and from simian immunodeficiency virus-infected macaques (5, 13). Only the sequence of E. bieneusi obtained from the pig is available (GenBank accession no. U61180) (5). This strain belongs to the type IV lineage, which was found in only 5% of our patients, but in none of these cases was contact with pigs documented. Furthermore, preliminary results of a recent study seemed to indicate the presence of E. bieneusi in surface water (19), a likely source of human contamination. Undoubtedly, the comparison of the types of E. bieneusi isolates from human, animal, and environmental sources will provide a better understanding of the epidemiology of E. bieneusi infections.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants from SIDACTION (Fondation pour la Recherche Médicale), Association des Professeurs de Pathologie Infectieuse et Tropicale (APPIT), Agence Nationale de Recherche sur le SIDA (ANRS 034, 035, and 054), and the Centre d'Etudes et de Recherches en Infectiologie.
We thank M. Rabodonirina for providing us with stool specimens from organ transplant recipients.
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FOOTNOTES |
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* Corresponding author. Mailing address: Clinique des Maladies Infectieuses, Hôpital Saint-Louis, 1 avenue C. Vellefaux, 75475 Paris Cedex 10, France. Phone: (33) 1 42 49 90 66. Fax: (33) 1 42 49 90 67. E-mail: molina{at}chu-stlouis.fr.
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References
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Journal of Clinical Microbiology, July 1998, p. 1882-1885, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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