Detection of Microsporidia in Travelers with Diarrhea

Abstract

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 l, 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-␤-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 [5Ј-TAATACGACTC ACTATAGGG-3Ј] and M-13 reverse primer [5Ј-TTCACACA GGAAACAGCTATGACG-3Ј]) flanking the cloned DNA fragments.

Detection and Identification of Enterocytozoon bieneusi and Encephalitozoon Species in Stool and Urine Specimens by PCR and Differential Hybridization

“…In comparison to microscopy with Uvitex 2B fluorescent stain, the spiking experiments showed a 10-to 1,000-foldhigher sensitivity of the PCR compared to microscopy, comparable to data from other studies (27,32,42). A further advantage of PCR over microscopy, although not detected in our study, could be the detection of infections by two (or more) different species in one patient (31,32). One clinical sample was positive by PCR, while it was negative by microscopy.…”

“…Enterocytozoon bieneusi and the Encephalitozoon species E. intestinalis, E. hellem, and E. cuniculi have been described as opportunistic pathogens in human immunodeficiency virus (HIV)-infected patients and other immunocompromised patients such as transplant recipients (11,18,24,29,34,36). Infections with microsporidia in immunocompetent individuals such as travelers have also been described (31,35). In HIV-infected patients, E. bieneusi and E. intestinalis can cause a severe, persistent diarrhea, and the species have frequently been isolated from stool specimens (11,16,17,34,39).…”

“…Furthermore, Encephalitozoon species are associated with rhinosinusitis, keratoconjunctivitis, nephritis, hepatitis, and disseminated infections (9,17,18,24,29). The Encephalitozoon species have been isolated from different clinical specimens such as urine and respiratory excretions (10,16,17), and E. hellem and E. cuniculi have occasionally been found in stool specimens (18,29,31).…”

Simultaneous Detection of Four Human Pathogenic Microsporidian Species from Clinical Samples by Oligonucleotide Microarray

“…Eleven of the positive samples contained E. intestinalis, and seven contained E. bieneusi, which are the most frequently microsporidian species detected in AIDS patients. Our data not only demonstrated the ability of microarrays to simultaneously detect multiple species but also further suggested that coinfection of microsporidia is not an uncommon event (24). However, it will be more interesting to investigate why this batch of archived samples had such a high incidence of dual infections.…”

Disseminated Microsporidiosis Caused by Encephalitozoon cuniculi III (Dog Type) in an Italian AIDS Patient: a Retrospective Study

“…The two other species of Encephalitozoon, E. cuniculi and E. hellem, are also known to cause disseminated infections including the urogenital, respiratory, and ocular organs. There are, however, two reports of enteric localization of E. cuniculi (2,5), and very recently, an E. hellem was recognized for the first time in stools of two immunocompetent travelers with diarrhea (6).…”