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Journal of Clinical Microbiology, April 2001, p. 1630-1632, Vol. 39, No. 4
Department of
Pediatrics1 and Department of Internal
Medicine I,3 University of Cologne, Cologne, and
Institut for Tropical Medicine, University of Tübingen,
Tübingen,2 Germany
Received 27 November 2000/Returned for modification 8 January
2001/Accepted 4 February 2001
We examined stool specimens of 148 returning travelers from an
outpatient department for tropical diseases for the appearence of
microsporidia using light microscopy and PCR. Intestinal
microsporidiosis was diagnosed for five patients by light microscopy
and for nine patients by PCR. Some cases were diagnosed only by PCR,
indicating that the true prevalence has to be determined by highly
sensitive techniques, such as PCR.
Diarrhea affects 20 to 70% of
travelers in tropical or subtropical countries. Typically,
self-limiting travelers' diarrhea is more of an inconvenience than a
life-threatening disease. In general, bacteria, e.g., Escherichia
coli, are the most common pathogens isolated in travelers'
diarrhea. However, different intestinal protozoan parasites are
recognized in travelers with diarrhea as well. Microsporidia are
important protozoan parasites in human immunodeficiency virus
(HIV)-infected patients. Limited data are available about
microsporidian infections in immunocompetent patients and travelers
(1, 17, 19, 21).
Stool samples were obtained from 148 travelers with diarrhea returning
from different tropical areas between January and March 1998. Stool
samples were examined by light microscopy (Uvitex 2B staining) and PCR
as previously described (16). PCR products were digested
with the restriction endonucleases PstI (20 U) and HaeIII (10 U) (Sigma) in a final volume of 15 µl,
producing distinctive fragments. The 250-bp DNA fragment of
Enterocytozoon bieneusi was not cut by PstI and
produced a 208-bp DNA fragment after digestion with HaeIII.
The 268-bp DNA fragment of Encephalitozoon cuniculi was cut
by PstI into two, 122- and 146-bp DNA fragments and by HaeIII into a 189-bp DNA fragment. The 270-bp DNA fragment
of Encephalitozoon intestinalis was cut by PstI
into two, 124- and 146-bp DNA fragments and by HaeIII into a
193-bp DNA fragment. The 279-bp DNA fragment of Encephalitozoon
hellem was cut by PstI into two, 133- and 146-bp DNA
fragments and by HaeIII into a 200-bp DNA fragment
(9). Purified DNA fragments were ligated into a pGEM-T
vector (Promega). Plasmids were transfected in competent E. coli cells (JM109) and plated on
ampicillin-isopropyl- Examination of stool samples from 148 returning travelers by light
microscopy showed microsporidian spores in stool samples of five
patients. One case was identified as E. bieneusi because of
the small size of the spores (2 µm). In all other cases, species differentiation was not possible. PCR amplification produced 250-bp DNA
fragments, indicating infection with E. bieneusi, from stool specimens of seven patients and from plasmids with the small-subunit (SSU) rRNA sequence of E. bieneusi. Four of these patients
were negative by light microscopy (Table
1). An approximately 270- to 280-bp DNA
fragment, indicating infection with an Encephalitozoon spp.,
was amplified from stool specimens of four patients and from plasmids
containing the SSU-rRNA genes of E. intestinalis, E. hellem,
and E. cuniculi (Table 1). In two specimens, 250-bp and 270- to 280-bp DNA fragments were amplified. Results of digestion with the
restriction endonucleases PstI and HaeIII and of
DNA sequencing are shown in Table 1. This confirms infection with E. bieneusi in five cases and double infection with E. bieneusi and E. hellem in two cases.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1630-1632.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Microsporidia in Travelers with
Diarrhea
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-D-thiogalactopyranoside-S - bromo - 4 - chloro - 3 - indolyl -
-D-galactopyranoside
agar plates for blue-white screening. White colonies were selected, and
plasmids were isolated from the cells by using the High Pure plasmid
isolation kit (Boehringer, Mannheim, Germany). Both DNA strands of the
cloned DNA fragments were sequenced on an automated DNA sequencer (ABI
Prism 377 DNA Sequencer; Applied Biosystems) using the Taq
FS BigDye-terminator cycle sequencing method starting with two
vector-directed primers (T7 primer [5'-TAATACGACTCACTATAGGG-3'] and M-13 reverse primer [5'-TTCACACAGGAAACAGCTATGACG-3'])
flanking the cloned DNA fragments.
TABLE 1.
Results of light microcopy, PCR amplification, digestion
of PCR products, and DNA sequencing of amplified PCR products from nine
patients with microsporidiosis
New pathogens continue to be discovered as the cause of travelers' diarrhea, and a few case reports have demonstrated that microsporidia may be the cause of travelers' diarrhea (1, 17, 19, 21). Human microsporidiosis has been reported predominantly from developed nations in North America, Europe, and Australia, but cases are now increasingly identified in the developing countries as well, and human infections with microsporidia have been reported from several African nations (2, 4, 6, 7, 11, 12, 14, 22), Southeast Asia (15), and Central and South America (3, 8, 24, 25). Although reliable estimates of the prevalence of microsporidiosis in developing countries are not available, some studies seem to indicate that the prevalence of intestinal microsporidiosis may be high in developing countries (10, 22). Most cases of human microsporidiosis are associated with immunosuppression, but increasing numbers of cases in non-HIV-infected immunocompetent patients are reported. These reports include infections of travelers to developing countries as well as infections of residents of various tropical countries (1, 5, 6, 11, 13, 14, 17, 19-21, 23). Only a few studies examined the role of microsporidia in returning travelers. One study examined stool samples of 750 Swedish travelers with and without diarrhea by light microscopy and identified only one case of intestinal microsporidiosis due to E. bieneusi (21). Another study examined 40 European travelers from the tropics with a clinical picture of protracted diarrhea and identified four cases of imported E. bieneusi infection: one HIV-infected short-term traveler, a pregnant long-term traveler, and two immunocompetent short-term travelers. Diarrhea was self-limited, and the spores cleared from the stools in all non-HIV-infected travelers but showed a chronic course in the HIV-infected one. Infections were diagnosed by light microscopy with confirmation by PCR (13). Spores of microsporidia were detected by light microscopy in the stools of four French travelers presenting clinically with chronic diarrhea. Molecular identification of microsporidian species was based on the PCR amplification of an SSU rRNA sequence followed by HinfI endonuclease restriction. E. intestinalis microsporidiosis was thus shown in two of the four patients examined (17). These three studies employed only light microscopy for detection of microsporidia in stools, and molecular-based techniques, such as PCR, were used only for confirmation and/or species differentiation. None of these studies used PCR for the examination of all stool samples. In our study, as well as in a previous study of members of our group (16), some of the cases were diagnosed only by PCR amplification. In a study that evaluated the prevalence of intestinal parasites in patients with diarrhea and AIDS in Zimbabwe, microsporidia were found by light microscopy in 10 out of 55 (18%), whereas PCR detected microsporidia in 28 out of 55 patients (51%) (10). Probably the limited sensitivity of light microscopy is responsible for these results. The detection limit of light microscopy has been determined to be between 104 and 106 microsporidian spores per g of stool, whereas PCR is able to detect spore concentrations as low as 102 per g of stool (16, 18). Thus the true prevalence of microsporidia has to be determined by highly sensitive techniques, such as PCR. However, diagnostic approaches regarding diarrhea are usually done late in the course of the disease, at a time when spore excretion in the stool may have decreased. In all of our cases except one, only low numbers of spores were seen by light microscopy. Interestingly, the two patients in our study with confirmed double infection due to E. bieneusi and E. hellem were a traveler and his daughter who returned from Singapore. In stool samples of the mother who accompanied the two patients to Singapore, no microsporidia were detected by light microscopy or by PCR. To the best of our knowledge this is the first report of E. hellem in stool samples and the first report of E. hellem in non-HIV-infected patients. Our study shows that intestinal microsporidiosis seems to be an underappreciated cause of travelers' diarrhea. New interest in these organisms by clinicians caring for travelers returning from tropical areas and improved diagnostic approaches should now help to increase our knowledge regarding the role of microsporidia in travelers' diarrhea.
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ACKNOWLEDGMENTS |
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We thank Britta Franzen for editorial assistance during preparation of the manuscript.
This work was supported by the Köln Fortune program from the Faculty of Medicine, University of Cologne.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Internal Medicine I, University of Cologne, Joseph-Stelzmann Str. 9, D-50924 Cologne, Germany. Phone: 49-(0)221-478-4433. Fax: 49-(0)221-478-5915. E-mail: Caspar.Franzen{at}Uni-Koeln.de.
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