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Previous Article | Next Article 
Journal of Clinical Microbiology, July 1999, p. 2317-2322, Vol. 37, No. 7
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Detection by an Immunofluorescence Test of Encephalitozoon
intestinalis Spores in Routinely Formalin-Fixed Stool Samples
Stored at Room Temperature
H.
Moura,1,2
F. C.
Sodre,1,3
F. J.
Bornay-Llinares,4
G. J.
Leitch,5
T.
Navin,1
S.
Wahlquist,1
R.
Bryan,1
I.
Meseguer,4 and
G.
S.
Visvesvara1,*
Division of Parasitic Diseases, National
Center for Infectious Diseases, Centers for Disease Control and
Prevention, Public Health Service, Department of Health and Human
Services,1 and Department of Physiology,
Morehouse School of Medicine,5 Atlanta,
Georgia; DPL, Faculdade de Ciências Médicas, Universidade
do Estado do Rio de Janeiro, and Hospital Evandro Chagas,
Instituto Oswaldo Cruz, FIOCRUZ, Rio de
Janeiro,2 and Departamento de Patologia,
Faculdade de Medicina, Universidade Federal Fluminense,
Niteroi,3 Brazil; and Unidad de
Biotecnología Microbiana, Centro de Bioinginiería,
Universidad Miguel Hernández, Elche, Alicante,
Spain4
Received 12 February 1999/Returned for modification 23 March
1999/Accepted 12 April 1999
 |
ABSTRACT |
Of the several microsporidia that infect humans,
Enterocytozoon bieneusi is known to cause a
gastrointestinal disease whereas Encephalitozoon
intestinalis causes both a disseminated and an intestinal
disease. Although several different staining techniques, including
the chromotrope technique and its modifications, Uvitex 2B, and the
quick-hot Gram-chromotrope procedure, detect microsporidian spores in fecal smears and other clinical samples,
they do not identify the species of microsporidia. A need for an
easily performed test therefore exists. We reevaluated
120 stool samples that had been found positive for microsporidia
previously, using the quick-hot Gram-chromotrope technique, and
segregated them into two groups on the basis of spore size. We also
screened the smears by immunofluorescence microscopy, using a
polyclonal rabbit anti-E. intestinalis serum at a dilution of 1:400. Spores in 29 (24.1%) of the 120 samples fluoresced brightly, indicating that they were E. intestinalis spores. No intense background or cross-reactivity
with bacteria, yeasts, or other structures in the stool samples was
seen. Additionally, the numbers of spores that fluoresced in seven of
these samples were substantially smaller than the numbers
of spores that were present in the stained smears, indicating that
these samples were probably derived from patients with mixed infections
of Enterocytozoon bieneusi and E. intestinalis. Because a 1:400 dilution of this serum does not
react with culture-grown Encephalitozoon hellem, Encephalitozoon cuniculi, or Vittaforma corneae
or with Enterocytozoon bieneusi spores in feces, we
concluded that an immunofluorescence test using this serum is
a good alternative for the specific identification of E. intestinalis infections.
| |
INTRODUCTION |
Microsporidia are ancient,
spore-forming, mitochondrion-lacking protozoa that are known to infect
patients with AIDS (8, 32, 44). Of the more than 1,000 species and as many as 100 genera of microsporidia, only 11 species included under 7 genera (i.e., Brachiola
vesicularum, Encephalitozoon cuniculi,
Encephalitozoon hellem, Encephalitozoon
intestinalis, Enterocytozoon bieneusi, Nosema
connori, Nosema ocularum, Pleistophora
sp., Trachipleistophora hominis, Trachipleistophora
anthropophthera, and Vittaforma corneae) are
known to cause infections in humans (7, 32, 36, 39, 44).
Enterocytozoon bieneusi is the most frequently identified microsporidial pathogen in fecal specimens from patients with AIDS.
Recently, however, it has also been found in respiratory samples from
two patients (14). E. intestinalis is the
second most frequently identified microsporidial pathogen in clinical specimens, including stool samples from AIDS patients (32). According to some reports, gastrointestinal disease caused by microsporidia accounts for 30% of diarrhea in patients with AIDS (32, 44). Although the chromotrope technique of Weber et al. (43) and modifications thereof (23, 31),
chemofluorescent agents such as Uvitex 2B (37), and the
recently developed quick-hot Gram-chromotrope procedure (27)
detect microsporidian spores in fecal smears and other clinical
samples, they do not identify the species of microsporidia. Polyclonal
and monoclonal antibodies (MAbs) have also been developed to identify
microsporidian spores in patient specimens, including fecal samples
(1, 2, 10, 19, 30, 33, 35, 40-42, 46, 47). However, with
the exception of an MAb to E. hellem (10,
41), none of these reagents can specifically identify particular
microsporidia. Definitive species identification can be accomplished by
electron microscopy (EM) (6, 8, 32, 44) or DNA-based
techniques (9, 11-13, 15, 17, 20, 21, 29, 40, 41), which
are not readily available to many clinical laboratories. There exists,
therefore, a need for a simple, easily performed, and reliable test to
identify E. intestinalis and Enterocytozoon
bieneusi, the two agents that have been found in fecal specimens
from patients with diarrhea. We have reported previously the
development of a polyclonal rabbit antiserum to E. intestinalis that identifies, based on an indirect immunofluorescence (IIF) technique, spores of E. intestinalis in Formalin-fixed smears of feces, urine, saliva, and
nasal secretions of a patient infected with E. intestinalis (40). We therefore screened a large number
of Formalin-fixed fecal samples, which had been found to be positive
for microsporidia by the chromotrope stain, with this antiserum in the
IIF test to identify the number and percentage of specimens that were
positive for E. intestinalis. Our goal was to develop a
diagnostic reagent for the routine identification of E. intestinalis in microsporidian-positive clinical specimens, including Formalin-fixed stool samples, that would be based on the use
of this serum. Our additional objectives were (i) to identify cross-reactions, if any, with culture-derived microsporidia, including E. cuniculi, E. hellem, E. intestinalis, and V. corneae, or Enterocytozoon bieneusi spores in Formalin-fixed fecal specimens; (ii) to
determine whether it is possible to specifically identify E. intestinalis spores in fecal samples that have been fixed in
Formalin for long periods of time; and (iii) to determine the
percentage of microsporidian-positive stool samples that harbor
E. intestinalis spores. The results of our
investigations are reported here.
| |
MATERIALS AND METHODS |
Fecal samples.
From 1991 to 1994 we monitored a cohort of
602 patients in Atlanta who were infected with human immunodeficiency
virus to determine the burden of disease associated with enteric
parasites (28). Forty-four (7.3%) of these patients were
found to have an intestinal infection with microsporidia on at least
one occasion. After the initial diagnostic tests on these patients were
completed, 35 had sufficient stool specimen remaining to allow us to
perform additional tests in an attempt to identify the particular
species of microsporidian. This report is based on an analysis of 120 specimens from these 35 patients. These samples had been stored at room
temperature (24°C) for about 3 to 5 years. Thin smears were made from
each of these unconcentrated samples, and the specimens were retested
for the presence of microsporidian spores by the quick-hot
Gram-chromotrope technique (27).
Since two sizes of spores were visualized during a cursory microscopic
examination of the stained smears, we selected 39 stained samples for
accurate measurements of the spores by using a computerized image
analyzer as well as a micrometer. At least 20 to 30 spores were
measured separately by two individuals (H.M. and G.S.V.) in order to
accurately determine the spore size.
EM.
Small aliquots of 10 of these stool samples were also
processed for EM. The stool samples were first washed three times by centrifugation in phosphate-buffered saline, and then the pellets were
resuspended in 2.5% glutaraldehyde buffered with 0.2 M cacodylate and
allowed to stand for 3 h. The samples were then postfixed with a
1% solution of OsO4, dehydrated in ethanol, embedded in Epon 812, and processed as described previously (40-42).
Reference isolates.
E. cuniculi (CDC:V282)
(13), E. hellem (CDC:0291:V213)
(41), E. intestinalis (CDC:V297)
(40), and V. corneae (34) were grown
in monkey kidney (E6) cell cultures as described previously. Spores
harvested from each of these cultures were also processed for IIF as
described previously (40-42).
IIF.
IIF was performed as described previously
(40-42). In brief, 10 µl of washed spores (5 × 106 per ml) from a culture was pipetted onto each well of
12-well coated slides; the slides were allowed to dry and stored at
20°C until used. Additionally, 5-µl aliquots of each of these 120 fecal specimens were pipetted onto individual wells of coated slides and allowed to dry. An IIF test was then performed with the
anti-E. intestinalis (CDC:V297) serum at a 1:400
dilution and with anti-rabbit immunoglobulin G conjugated with
fluorescein isothiocyanate as described previously (40).
Evan's blue was included as a counterstain. Prior to screening the
stool smears with the anti-E. intestinalis (CDC:V297)
serum, we titrated the serum by using several dilutions, starting with
1:100 and ending with 1:3,200, against cell culture-derived spores of
E. cuniculi, E. hellem, E. intestinalis, and V. corneae along with a stool sample
that was previously found to be positive for E. intestinalis by EM (40) as well as a stool sample that had been identified as positive for Enterocytozoon bieneusi
by PCR (11).
| |
RESULTS |
Quick-hot Gram-chromotrope staining.
Microsporidian spores were detected in smears made from all of
the 120 fecal samples that were also positive earlier. The spores
stained dark violet and exhibited at least one of the characteristic features of a spore, i.e., an equatorial belt-like stripe, a vacuole, and/or the presence of gram-positive granules (Fig.
1a).
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FIG. 1.
(a) Fecal smear stained by the Gram-chromotrope
procedure. Note the two sizes of spores in the inset (larger spores at
arrows). Magnification, ×1,250. (b) Spores in the fecal smear reacted
with the anti-E. intestinalis serum at a 1:400 dilution
and exhibited bright fluorescence. Note the nonspecific reaction of
diplococcus-like and fungal organisms in the smear that can be easily
distinguished from the specific fluorescence patterns of the brightly
fluorescing spores. Magnification, ×500.
|
|
The average diameter of the spores in 11 (IIF negative) of the 39 samples that were selected for measurement was 1.43 µm (range, 0.87 to 1.98 µm). In 21 of the samples that were IIF positive, however,
the spores had an average measurement of 1.59 µm (range, 0.95 to 3 µm); a few, however, measured around 1 µm (Fig. 1a). In the
remaining seven samples suspected to contain both Enterocytozoon bieneusi and E. intestinalis, the spores had an
average measurement of 1.56 µm (range, 0.80 to 2.56 µm).
IIF.
To ascertain the cross-reactivity of the
anti-E. intestinalis serum with spores of E. intestinalis and Enterocytozoon bieneusi in stool
smears and with spores of culture-grown microsporidia (E. cuniculi, E. hellem, E. intestinalis, and V. corneae), we allowed the spores of
different microsporidia to react with different dilutions of the serum,
starting with the 1:100 dilution. Spores of V. corneae and
Enterocytozoon bieneusi showed no fluorescence even at a
serum dilution of 1:100. Since spores of E. cuniculi and E. hellem showed some cross-reactivity at the lower
dilutions (Table 1), we elected to use a
1:400 dilution to screen fecal samples.
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TABLE 1.
Reactivities of the different dilutions of the
anti-E. intestinalis serum with spores of selected
microsporidial species
|
|
In the IIF assay, spores in 29 (24.1%) of 120 smears reacted with the
anti-E. intestinalis serum and produced bright
fluorescence (Fig. 1b), indicating that they were E. intestinalis spores. Although in some samples diplococcus-like
bacterial spores and fungal organisms exhibited dull fluorescence, they
could very easily be distinguished from the microsporidian spores with
the bright fluorescence (Fig. 1b). The numbers of spores that
fluoresced in 7 of the 29 samples were relatively low compared
with those in the corresponding Gram-chromotrope-stained smears of
these samples, indicating that these specimens may have contained two
different microsporidia, namely E. intestinalis and
Enterocytozoon bieneusi.
The 35 patients each had between 1 and 16 stool specimens that
were positive for microsporidia. Twenty-one (60.0%) of the patients
were infected only with Enterocytozoon bieneusi, and four
(11.4%) were infected only with E. intestinalis.
Six patients had at least one stool specimen with both
E. intestinalis and Enterocytozoon bieneusi,
and four patients had E. intestinalis on at least one
occasion and Enterocytozoon bieneusi on at least one
separate occasion.
EM.
Among the 10 samples that were examined by EM,
microsporidia characteristic of Enterocytozoon bieneusi
(Fig. 2a and b) were observed in only
five, none of which was positive in the IIF assay; 3 samples, all of
which were positive in the IIF assay, had spores characteristic of
E. intestinalis (Fig. 2c), while 2 samples, both
positive in the IIF assay, had spores of both
Enterocytozoon bieneusi and E. intestinalis. E. intestinalis spores, however, were in
small numbers, as visualized by the IIF assay. Enterocytozoon bieneusi spores could be easily identified on the basis of their smaller size (~1 µm) and double rows of polar tube coils.
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FIG. 2.
(a) Electron micrograph of a fecal sample showing an
Enterocytozoon bieneusi sporoblast with polar tube coils in
double rows (at arrows); bar = 500 nm. (b) A mature spore of
Enterocytozoon bieneusi with the characteristic double turns
of the polar tubule (at arrow); bar = 500 nm. (c) Electron
micrograph of a fecal sample showing an E. intestinalis
spore. Note the polar tubule coils (arrows). Bar = 500 nm.
|
|
| |
DISCUSSION |
Identification of an agent of microsporidiosis in general, and
gastroenteral microsporidiosis in particular, to the species level is
important, since infections with E. intestinalis can be
successfully treated with albendazole whereas those caused by
Enterocytozoon bieneusi may be refractory to
albendazole therapy (32, 44). Identification to the species
level, however, can be difficult and require time-consuming and
specialized techniques, including EM and PCR, which not only are
tedious and time-consuming but also are currently available only in a
few laboratories. It is therefore advantageous to develop
species-specific serologic reagents that can be used for the
identification of the species that causes the disease so that specific
therapy can be instituted.
According to several reports, both polyclonal and monoclonal reagents
have been developed to identify microsporidia in fecal specimens; none
of them, however, can unequivocally identify microsporidia to the
species level, since cross-reactivity of these reagents with other
microsporidia of clinical importance has been demonstrated (1, 19,
46, 47). According to a recent report, two MAbs identified (for
up to only 6 weeks) E. intestinalis spores in fresh
stool specimens that were refrigerated but not in routinely Formalin-fixed stool samples (2). Another recent report,
which discusses an anti-E. intestinalis antibody
produced in BALB/c mice, states that the antibody can identify
E. intestinalis spores in fresh stool samples fixed in
ice-cold acetone but not in Formalin-fixed specimens (30).
We have also reported previously on the development of a polyclonal
anti-E. intestinalis (CDC:V297) serum that can be used to identify E. intestinalis spores in clinical
specimens, including Formalin-fixed stool samples (40).
Further, by using the CDC:V297 serum and flow cytometry, we have
recently shown that both Formalin-treated and untreated spores of
E. cuniculi, E. hellem, and
E. intestinalis could be clearly identified to the
species level by analyzing the gated data on light-scatter profiles and
fluorescence histograms (26). In the present
investigation, we examined the specificity of the serum by reacting it
with spores of E. hellem, E. cuniculi, E. intestinalis, and V. corneae obtained
from in vitro culture as well as with spores in two
Formalin-fixed stool samples (which served as positive
and negative controls) as well as 120 Formalin-fixed stool samples that
had been stored at room temperature for 3 to 5 years. The results of
these experiments clearly indicated that this rabbit anti-E.
intestinalis serum, at a dilution of 1:400, specifically
identified E. intestinalis spores from cell culture and
in some of the Formalin-fixed stool specimens and produced bright apple green fluorescence. It did not cross-react with
E. cuniculi, E. hellem, or V. corneae from culture or with Enterocytozoon bieneusi
from stool specimens (35). These results clearly
indicate that E. intestinalis spores can be
identified even in those samples fixed in formalin and stored at
room temperature for up to 5 years. Hence, an antibody like this, if
commercially available, would be very useful for screening
microsporidian-positive samples for the specific identification of
E. intestinalis spores even when they are present in
small numbers.
In a previous study, based on EM and PCR of intestinal biopsy samples
from patients with diarrhea, an overall rate of 44.1% infection with
microsporidia (36.8% with Enterocytozoon bieneusi and 7.3%
with E. intestinalis) was recorded (9). In
another study, based on PCR of stool samples, a 10% incidence of
E. intestinalis was found (24). According to
a third study, based on a PCR assay, the prevalence of infection with
E. intestinalis is probably higher than previously
reported, because of latent infection (20). It is
interesting that in the present study, spores in 29 (24.1%) of the
120 fecal specimens tested reacted intensely with the
anti-E. intestinalis serum and produced a bright
fluorescence. Since it was not possible to perform EM on all of the 120 specimens because of time, personnel, and cost constraints, we elected
to carry out EM on 10 selected samples. The EM results for these 10 specimens confirmed that 5 of these samples that were negative in the
IIF assay had only spores characteristic of Enterocytozoon
bieneusi, 3 samples that were positive in the IIF test had only
spores characteristic of the genus Encephalitozoon, and the
2 samples that were positive in the IIF test had spores that were
characteristic of both Enterocytozoon bieneusi and
Encephalitozoon spp. The number of spores that fluoresced positively with the anti-E. intestinalis serum was,
however, small compared with the total number of spores in the stained
smears. These results, albeit based on limited comparisons, indicate
that the reagent is specific and sensitive.
Microscopic examination of the stained smears of these 29 fecal samples
revealed that a majority of spores appeared to be 1.59 µm in diameter
(range, 0.95 to 3 µm), which is comparable to the size of
E. intestinalis spores (2, 6, 8, 16, 18, 30, 32,
38, 40, 42). On reevaluation, however, 7 of these 29 stool
samples appeared to contain spores of two different sizes, namely ~1
µm and 
2 µm. Enterocytozoon bieneusi spores are known
to be ~1 µm in diameter, whereas those of E. intestinalis measure ~2 µm or greater; further, the number of spores that fluoresced was considerably smaller than that in the Gram-chromotrope-stained smears. Based on these two observations, we
concluded that these samples were positive for both E. intestinalis and Enterocytozoon bieneusi spores,
indicating that the persons from whom the samples were obtained had
mixed infections. Mixed infections with E. intestinalis
and Enterocytozoon bieneusi have been reported previously
(4, 38).
It is well known that E. cuniculi infects a variety of
animals, including humans (8, 13, 15, 17, 22, 32, 44, 45).
Enterocytozoon bieneusi was recently identified for the first time in fecal specimens from domestic pigs (15) and in simian immunodeficiency virus-inoculated macaques with hepatobiliary disease (25). E. hellem has also been
identified in birds (budgerigars) for the first time (3).
Further, we have recently identified spores of E. intestinalis in Formalin-fixed fecal specimens of domestic animals
(i.e., pigs, a dog, a cow, a goat, and a donkey) by several
techniques, including EM, PCR, and IIF (5). For the IIF
assay, we used our anti-E. intestinalis serum at a
1:400 dilution as discussed here. The EM and, more importantly,
the PCR data agreed with the immunofluorescence results
(5). These reports therefore underscore the urgent need
for the development of a simple, reliable, noninvasive technique
for the identification of these microsporidia in fecal and
environmental samples. Our study results are a first step in the
development of species-specific reagents that can identify
E. intestinalis in fecal specimens and should provide
impetus for others to develop simple and reliable species-specific
reagents to identify other microsporidia in stool samples as well as in
the environment. Additionally, species-specific reagents would be
invaluable not only in the diagnosis of microsporidiosis, so that
interdiction through chemotherapeutic agents would be possible, but
also in the identification of specific agents in animal and
environmental samples and epidemiologic investigations.
| |
ACKNOWLEDGMENTS |
H. Moura and F. C. Sodre were recipients of scholarships
from Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico, CNPq, Brazil. G. J. Leitch was supported in
part by Public Health Service grant RR03034. F. J. Bornay-Llinares
was supported in part by a grant from the Sociedad Española de
Microbiología Cliníca y Enfermedades Infecciosas
(SEIMC)-Bayer 1996.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Parasitic Diseases, M.S.-F-13, Centers for Disease Control and
Prevention, 4770 Buford Hwy. NE, Atlanta, GA 30341-3724. Phone: (770)
488-4417. Fax: (770) 488-4253. E-mail: GSV1{at}CDC.GOV.
| |
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Journal of Clinical Microbiology, July 1999, p. 2317-2322, Vol. 37, No. 7
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Abstract
Introduction
