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Journal of Clinical Microbiology, January 1998, p. 37-40, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Encephalitozoon
intestinalis in Travelers with Chronic Diarrhea by Specific
PCR Amplification
Laurent
Raynaud,1,2
Frederic
Delbac,3
Veronique
Broussolle,3
Meja
Rabodonirina,2
Veronique
Girault,2
Martine
Wallon,2
Gregoire
Cozon,4
Christian P.
Vivares,3 and
François
Peyron2,*
ESSA, 69500 Bron,1
Département de Parasitologie, Faculté de
Médecine, Université Claude Bernard, 69373 Lyon Cedex
08,2
Protistologie Moléculaire et
Cellulaire des Parasites Opportunistes, LBCP, ESA CNRS 6023,
Université Blaise Pascal, 63177 Aubière
Cedex,3 and
Service Immunologie,
Hôpital de la Croix Rousse, 69317 Lyon Cedex
04,4 France
Received 31 March 1997/Returned for modification 16 September
1997/Accepted 10 October 1997
 |
ABSTRACT |
With the use of Weber's modified trichrome and Uvitex 2B
techniques, spores of microsporidia were detected in the stools of four
travelers presenting clinically with chronic diarrhea. The general
health of these patients was not impaired, and human immunodeficiency virus screening was negative. Immune evaluation, including the study of
lymphocytic subpopulations, assay of serum immunoglobulins, and an
intradermal multitest, showed normal results. Molecular identification
of microsporidian species was based on the PCR amplification of a
small-subunit rRNA sequence followed by HinfI endonuclease
restriction. Encephalitozoon intestinalis microsporidiosis was thus shown in two of the four patients examined. In two patients, therapy based on albendazole made stools devoid of microsporidian spores without influence on the intestinal disorders. The pathogenic role of E. intestinalis in immunocompetent individuals
remains to be demonstrated.
| |
INTRODUCTION |
Microsporidia are ubiquitous
intracellular parasitic protozoa affecting the whole animal kingdom
(4). Seven genera are pathogenic in humans:
Encephalitozoon, Enterocytozoon,
Nosema, Pleistophora, Vittaforma,
Trachypleistophora, and Microsporidium, the
latter including all species with undetermined status. Some of them are
known to be opportunistic pathogens in immunodepressed patients
(8). They may cause chronic diarrhea with food
malabsorption, as well as disseminated impairments (2, 3,
7). Ocular and gastrointestinal failures related to microsporidia
have also been described in immunocompetent ones (1, 5, 10, 14, 17, 18). Staining methods (19, 22) and
immunodiagnostic tests (15, 20, 21, 23, 24) can
differentiate microsporidia from bacteria and yeasts in clinical
samples such as stool samples, but precise identification of the
species involved is not always successful. Recently, PCR amplification
of conserved ribosomal DNA (rDNA) sequences was used to detect
intestinal microsporidia in biopsy and stool specimens (6, 9, 11,
13, 16).
We screened four human immunodeficiency virus (HIV)-negative travelers
with chronic diarrhea for microsporidia and gave them a complete
immunologic evaluation. After detection of spores in stool samples by
light microscopy, we tried to identify the corresponding parasite
species. However, electron microscopy failed to provide evidence of
microsporidia and no significant results were obtained for the
detection of Encephalitozoon cuniculi by Western blotting and enzyme-linked immunosorbent assay. Thus, we decided to develop a
new PCR procedure ensuring the differentiation of any known microsporidian species pathogenic to humans. This procedure enabled us
to identify Encephalitozoon intestinalis in two
immunocompetent patients.
| |
MATERIALS AND METHODS |
Patients.
The patients were travelers presenting with
chronic diarrhea. When microsporidian spores were detected in stool
samples as judged by Weber's modified trichrome (22) and
Uvitex 2B (19) techniques, an immunologic evaluation was
performed including HIV tests, assay of immunoglobulins, and a study of
lymphocytic subpopulations by flow cytometry after triple labeling with
anti-CD3, -CD4, -CD8, -CD56 (NK cells), and -DC19 (B lymphocytes)
antibodies, and an intradermal multitest (Bio-Mérieux, Marcy
l'Etoile, France). Treatment with albendazole 400 mg twice daily was
prescribed for 20 days, and the patients were re-examined 1 month after
the end of the treatment.
Clinical samples from two homosexual AIDS patients (<20
CD4+ cells per µl) with intestinal microsporidiosis were
used as a positive control.
Stool samples and parasite cultures.
Formalin-fixed stool
samples were washed several times in phosphate-buffered saline (PBS)
and stored at 4°C. Septata intestinalis Cali, Kotler, and
Orenstein 1993 (2), subsequently reclassified as
Encephalitozoon intestinalis by Hartskeerl et al.
(12); E. cuniculi; and E. hellem were
grown in vitro in MRC-5 human lung fibroblasts (Bio-Mérieux) or
Madin-Darby canine kidney (MDCK) cells (Bio-Whittaker) in
75-cm2 tissue culture flasks (Polylabo) containing minimum
essential medium supplemented with L-glutamine, 5% fetal
calf serum, and diverse antibiotics (ampicillin, penicillin, and
streptomycin). The cell cultures were incubated at 37°C with 5%
CO2 in an air atmosphere. Supernatants containing mature
spores were collected; spores were then sedimented by centrifugation,
washed, and stored in 0.1 M PBS (pH 7.4) at 4°C.
DNA extraction.
Stool specimens were mixed with 1 volume of
PBS buffer and then centrifuged at 18,000 × g for 2 min. The pellets were washed in PBS and resuspended in 1 ml of 1%
sodium dodecyl sulfate-300 mM Tris (pH 9.0)-100 mM EDTA. After
incubation at 65°C for 30 min, suspensions were centrifuged and
resuspended in 500 µl of lysis buffer (10 mM Tris, 100 mM NaCl, 1 mg
of proteinase K [Sigma] per ml, 200 U of Lyticase [Sigma]).
Mechanical disruption was performed with zirconium beads (0.1-mm
diameter; Biospec Products Inc., Bartlesville, Okla.). Following
addition of 2% sodium dodecyl sulfate and 1 mg of proteinase K per ml,
extracts were incubated at 55°C for 3 h and then proteins were
precipitated with 1 M potassium acetate for 1 h at 4°C. DNA was
phenol-chloroform extracted, precipitated with ethanol for 1 h,
and resuspended in 50 µl of sterile water.
E. cuniculi, E. hellem, and E. intestinalis spores collected from MRC-5 or MDCK cell cultures
were boiled at 100°C for 10 min to release DNA.
PCR amplification.
Primers for PCR were chosen to amplify a
conserved region of the small-subunit (SSU) rRNA gene of four
microsporidia reported in AIDS patients: E. cuniculi,
E. hellem, E. intestinalis, and Enterocytozoon bieneusi. Forward primer C1
(5'-CACCAGGTTGATTCTGCC-3') and reverse primer C2
(5'-GTGACGGGCGGTGTGTAC-3') were determined by GenBank
sequence analysis of these species. C1 was complementary to bases 1 to
18 of each one, C2 was complementary to bases 1169 to 1186 of E. intestinalis (accession no. U09929), bases 1173 to 1190 of
E. cuniculi (accession no. L17072), bases 1188 to 1205 of
E. hellem (accession no. L19070), and bases 1152 to 1170 of
E. bieneusi (accession no. L16868).
Amplification was done in a 50-µl reaction mixture including 12.5 pmol of each primer, 200 µM each deoxynucleoside triphosphate,
2 mM
MgCl
2, and 1 U of
Taq DNA polymerase (Goldstar,
Eurogentec
Belgium). Two different volumes of each DNA preparation were
regularly
tested: 1 µl of the initial extract and 1 µl of the
extract diluted
with 9 volumes of sterile water.
After denaturation of the DNA at 94°C for 5 min, 30 cycles were run
with a Techne PHC-3 apparatus as follows: denaturation
at 94°C for 1 min, annealing at 56°C for 1 min, and elongation
at 72°C for 1 min,
with 5 min of extension at 72°C after the 30
cycles. Amplified
products were electrophoretically analyzed on
agarose gel and stained
with ethidium bromide.
Digestion of PCR products.
Restriction endonucleases
HindIII and HinfI (Eurogentec) were used to
digest amplified fragments obtained from culture and stool DNA
extracts. After amplification in a 50-µl reaction mixture, DNA was
precipitated in 100 µl of ethanol with 0.1 M NaCl and then
resuspended in 10 µl of sterile water. Three microliters was digested
with 3 U of HindIII or 5 U of HinfI in a
final volume of 10 µl. Digest fragments were electrophoretically
analyzed on agarose gel and stained with ethidium bromide.
| |
RESULTS |
Development of a PCR protocol.
A PCR amplification procedure
has been developed to facilitate the detection of microsporidia in
human samples. The primers were designed to amplify a large part of the
16S rDNA from four microsporidian species, including the two major
species involved in intestinal diseases: E. bieneusi
(1,170-bp amplicon) and E. intestinalis (1,186-bp amplicon).
When DNAs extracted from spores of E. cuniculi, E. hellem, and E. intestinalis cultures were used, agarose
gel electrophoresis revealed the amplification of a DNA fragment with a
size of about 1,200 bp (Fig. 1, lane 1).
As also expected, the 1,200-bp PCR product was found in fecal samples from two AIDS patients, SID1 and SID2 (Fig. 1, lanes 6 and 7).
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FIG. 1.
Analysis of PCR products by 1.5% agarose gel
electrophoresis. A 1,200-bp DNA fragment of the SSU rRNA gene of
microsporidia was amplified by using primers C1 and C2. Molecular size
markers (lane m) were  phage DNA digested with EcoRI and
HindIII. DNAs were extracted from E. intestinalis in MRC-5 cell cultures (lane 1, positive control),
immunocompetent patient (IC1, IC2, IC3, and IC4) stools (lanes 2, 3, 4, and 5), HIV-infected patient (SID1 and SID2) stools (lanes 6 and 7),
and a negative control (lane 8).
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|
The amplified rDNA sequence displays one
HindIII
restriction site in
E. bieneusi and none in any
Encephalitozoon species.
Thus, digestion of amplified
products with the restriction endonuclease
HindIII may
be used to distinguish between the two candidate genera
for intestinal
infection. Results of digestion experiments are
shown in Fig.
2. The PCR product from each of the two
AIDS patients
was cleaved by
HindIII, giving two
fragments with sizes close
to those expected: 784 and 386 bp (Fig.
2,
lanes 5 and 6). This
indicated that these patients were infected with
E. bieneusi,
which is in agreement with some electron
microscopy observations
(data not shown).
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FIG. 2.
Digestion of PCR products (1,200-bp DNA fragment) by
HindIII restriction endonuclease. Shown is a 1.5%
agarose gel containing E. intestinalis rDNA amplified before
(lane 1) and after (lane 2) HindIII digestion and
digested, amplified fragments from patients IC1 and IC3 (lanes 3 and 4)
and patients SID1 and SID2 (lanes 5 and 6). Lane 7 contained a negative
control. Two bands were obtained from E. bieneusi-infected
patients (800 and 400 bp). Amplified DNA fragments of E. intestinalis, E. cuniculi, and E. hellem
were not cleaved by HindIII. Lane m contained the same
molecular size markers as lane m in Fig. 1.
|
|
To identify
Encephalitozoon species,
HinfI
digestion of the 1,200-bp amplicons was performed. From the examination
of sequences
deposited in GenBank, it could be predicted that these
species
may be differentiated on the basis of the number of
HinfI restriction
sites: one for
E. cuniculi, two
for
E. hellem, and three for
E. intestinalis,
excluding one site at position 10 within each amplicon.
Species-specific restriction patterns were indeed observed, the
sizes
of the different fragments being 350 and 830 bp for
E. cuniculi (Fig.
3, lane 2); 120, 250, 350, and 460 for
E. intestinalis (lane
3); and 260, 350, and
580 for
E. hellem (lane 4). Digestion with
the same enzyme
of 1,200-bp amplicons from the two AIDS patients
produced two
distinctive DNA fragments with sizes (230 and 940
bp; Fig.
3, lanes 7 and 8) different from those of
E. cuniculi (350 and 830 bp).
It was confirmed that the two immunocompromised
patients were infected
with
E. bieneusi, the amplified rDNA sequence
of which
displays one
HinfI restriction site, at position 238.
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FIG. 3.
Digestion of a 1,200-bp amplicon by restriction
endonuclease HinfI. Shown is a 2% agarose gel. Lanes: 1, 1,200-bp PCR product before HinfI digestion; 2, E. cuniculi; 3, E. intestinalis; lane 4, E. hellem; 5 and 6, patients IC1 and IC3; 7 and 8, patients SID1 and
SID2. Lane m contained the same molecular size markers as lane m in
Fig. 1.
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|
Diagnosis of diarrheic travelers.
For this study, four
travelers were selected. They were males with a mean age of 29 years
and had had diarrhea for 1 to 71 months. They had traveled in Africa,
Nepal, or Southeast Asia. The mean daily number of stools was four,
they contained neither blood nor mucus, and the patients' general
health was good. Only one patient complained of nonsystematic abdominal
pain. The four travelers (IC1, IC2, IC3, and IC4) were HIV negative.
The test for lymphocytic subpopulations and the cutaneous
multitest yielded normal results (Table
1). Immunoglobulin levels in serum were normal, except in the patient with protracted diarrhea (6 years), whose
immunoglobulin A (IgA) concentration was 0.86 g/liter (normal, 1 to 3 g/liter). An assay of the IgA in the saliva of this patient yielded 100 mg/liter (normal range, 87 to 500 mg/liter). Stool examinations using Weber's modified trichrome and Uvitex 2B
techniques revealed the presence of microsporidian spores. The patients
were also screened for parasites by direct examination, culture of stools, and a Ziehl-Neelsen test for cryptosporidia, and no evidence of
eukaryotic pathogens other than microsporidia was obtained.
The above-described PCR amplification procedure was applied to stool
samples from four nonimmunocompromised patients. A positive
response
was obtained with samples from only patients IC1 and
IC3 (Fig.
1, lanes
2 and 4), suggesting a microsporidial infection.
HinfI
digestion showed the four bands which are relatable to the
presence of
E. intestinalis (Fig.
3, lanes 5 and 6). The stool
samples
from patients IC2 and IC4 were spiked with cultured
E. cuniculi spores. The expected amplified products were obtained,
the detection limit of particular samples varying between 20 and
100 spores per 0.1 g of stool. Repeated sampling provided the
same
results.
| |
DISCUSSION |
Our PCR method involves the digestion of an amplified product as
described by Fedorko et al. (9), except that the product corresponds to a much larger fragment of the SSU rRNA gene (more than
90% of the whole length). Ombrouck et al. (16) have
detected E. intestinalis in stool samples from AIDS patients
by amplifying a 380-bp DNA fragment with a specific reverse primer.
Since it has been recently shown that E. cuniculi and
E. hellem spores could be present in stools of AIDS patients
(6), the differentiation of Encephalitozoon
species is essential. This has been achieved through HinfI
digestion of the 1,200-bp amplicon, which provides specific banding
patterns. E. bieneusi and the three
Encephalitozoon species can be specifically identified. The
data support an E. intestinalis infection in two
nonimmunocompromised humans (IC1 and IC3). Our DNA extraction and
analysis protocol for stool samples is realizable in 1 day. This time
could be shortened if a heating procedure were used for cell lysis
(16).
Microsporidial pathogenicity in immunocompetent patients is still
poorly understood, and difficulties in diagnosis persist. In the
patients examined here, the Uvitex 2B and Weber staining methods
(19, 22) provided evidence of microsporidian infection independent of the HIV syndrome. An IgA deficit can be ruled out in our
patients. This deficit occurs frequently, and it may be asymptomatic
and detected at a late stage of disease. IgA plays an important role in
mucosal immunity, especially in the intestine, so that a partial
secretion deficit might promote the development of microsporidia. In
the literature, we found several reported cases of intestinal
microsporidia in the absence of immunodeficiency proven on the basis of
negative HIV tests (1, 10, 14, 17, 18). E. bieneusi was the predominant species, causing diarrhea that
subsided spontaneously in less than 6 weeks. One single case of
E. intestinalis infection concerned a homosexual patient
whose mate was also a carrier of the parasite (10).
Two diarrheic travelers (IC1 and IC3) were found to be microsporidium
positive by staining methods and PCR amplification. Albendazole
treatment led to the elimination of E. intestinalis spores
in stools (checked on three occasions in a 4-month period). However, as
the clinical signs persisted, the pathogenic role of E. intestinalis is debatable.
Amplification failed to detect microsporidia in patients IC2 and IC4,
while staining methods gave positive responses. Two interpretations may
be considered: (i) the stool samples contained some DNA polymerase
inhibitors, or (ii) the quantity of spores was below the limit of
detection. As shown by Katzwinkel-Wladarsch et al. (13) with
another PCR method applied to stools, the detection limit varies
between 3 and 100 spores per 0.1 g of stool, depending on the
particular samples, which may have qualitative and quantitative differences in DNA polymerase inhibitors. Results of our experiments involving the addition of cultured parasites to negative stool samples
suggest that the concentration of microscopically identified microsporidian spores was probably below 20 spores per 0.1 g of stool. Patient IC4 was treated with albendazole. The clinical signs
subsided, and microsporidium-like spores were still detected with
Uvitex 2B. Thus, the hypothesis that another pathogenic organism was
responsible for the chronic diarrhea cannot be excluded.
In conclusion, although microsporidia in stool samples can be easily
detected by staining procedures, our PCR protocol should be of interest
for the rapid determination of any particular species.
| |
ACKNOWLEDGMENTS |
L. Raynaud and F. Delbac contributed equally to this study.
We thank Elizabeth U. Canning for providing reference isolates of
E. cuniculi and E. hellem and T. Van Gool for
providing a reference isolate of E. intestinalis. We are
grateful to Guy Métenier for helpful discussions and comments on
the manuscript.
This work was supported by a grant from SIDACTION 1995-96.
Véronique Broussolle was supported by a SIDACTION postdoctoral scholarship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Département de Parasitologie, Faculté de Médecine,
Université Claude Bernard, 8 Avenue Rockefeller 69373 Lyon Cedex
08, France. Phone: 33-04-78-77-70-00. Fax: 0033-04-78-75-17-72. E-mail:
peyron{at}cismsun.univ-lyon1.fr.
| |
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Journal of Clinical Microbiology, January 1998, p. 37-40, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
