Relevant Criteria for Detecting Microsporidia in Stool Specimens

By using different staining techniques, 479 stool specimens from 212 diarrheic patients with AIDS were examined for microsporidian spores. Calcofluor fluorescence staining of 119 specimens revealed fluorescent ovoid structures of microsporidian size. Staining of these samples according to the method of Weber et al. (R. Weber, R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin, and G. S. Visvesvara, N. Engl. J. Med. 326:161–166, 1992) with trichrome produced six specimens with pinkish spores containing the characteristic microsporidian belt-like structure. The 6 specimens were processed for transmission electron microscopy, as were another 21 specimens which did not present the belt-like structure after trichrome staining but which looked highly suspicious after fluorescence staining. In these 21 samples, only fungal spores and, particularly, bacterial Clostridium spores were demonstrated, whereas in the 6 samples diagnosed positive after trichrome staining, the existence of microsporidia could be verified by electron microscopy. Based on our observations, we propose that the belt-like structure seen with the Weber stains in microsporidian spores corresponds to structures existing in priming-stage spores. The results suggest that routine microscopical fecal diagnosis for microsporidian infection should include a screening by fluorescence staining and, subsequently, a confirmatory viewing of fluorescence-positive samples after trichrome staining.     

Comparison of three staining methods for detecting microsporidia in fluids.

Calcofluor white 2MR, modified trichrome blue, and indirect immunofluorescent antibody (IFA) staining methods were evaluated and compared for detecting microsporidia in stool. Serial 10-fold dilutions of Encephalitozoon (Septata) intestinalis were prepared in three formalinized stool specimens or in Tris-buffered saline. Ten-microliter aliquots were smeared onto glass slides, fixed with methanol, stained, and read by at least three individuals. The results indicated that the calcofluor stain was the most sensitive method, required approximately 15 min to perform, but did generate some false-positive results due to similarly staining small yeast cells. The modified trichrome blue stain was nearly as sensitive as the calcofluor stain and allowed for easier distinction between microsporidia and yeast cells. This stain, however, required approximately 60 min to perform. The IFA stain with polyclonal murine antiserum against E. intestinalis was the least sensitive of the methods and required approximately 130 min to perform. The lower limit of detection with the calcofluor and modified trichrome stains was a concentration of about 500 organisms in 10 microliters of stool to detect one microsporidian after viewing 50 fields at a final magnification of x1,000. Reliability was also addressed by use of 74 stool, urine, and intestinal fluid specimens, 50 of which were confirmed for the presence of microsporidia by transmission electron microscopy (TEM). All TEM-positive specimens were detected by calcofluor and modified trichrome blue staining. Ten specimens were not detected by the IFA stain. An additional seven TEM-negative specimens were read positive for microsporidia with the calcofluor stain, and of these, five also were read positive with the modified trichrome blue stain. The resulting diagnostic paradigm was to screen specimens with the calcofluor stain and to confirm the results with the modified trichrome stain. IFA, which was less sensitive, may become useful for microsporidian species identification as specific antibodies become available.     

Modified technique to recover microsporidian spores in sodium acetate-acetic acid-formalin-fixed fecal samples by light microscopy and correlation with transmission electron microscopy.

Microsporidia are an emerging cause of significant disease, particularly in the immunocompromised host. Until recently, the diagnosis of enteric infections has required invasive sampling, the use of expensive technology, and considerable technological expertise. The purpose of the present study was to examine three modifications to the processing of fecal specimens for light microscopy (LM) examination for microsporidian spores: the use of pretreatment with potassium hydroxide, modified centrifugation conditions, and a modified staining technique. A sodium acetate-acetic acid-formalin-fixed fecal sample containing numerous microsporidian spores confirmed to be positive by transmission electron microscopy (TEM) was used in all studies performed. A simulation of a heavy to lightly infected individual was used. The results of LM were correlated with those of TEM. Duplicate smears were stained with Weber's modified trichrome and Giemsa (GS) stains. The stained slides were randomized and examined blindly by LM at x 625 and x 1,250 magnifications. A portion of the dilutions after centrifugation were fixed for TEM. The Weber modified trichrome stain performance rating was higher than the Giemsa stain rating because of ease of interpretation, and material stained with Weber modified trichrome stain required less examination time at a lower magnification. The number of positive smears and the quantity of spores detected were significantly higher following pretreatment of the sample with KOH. TEM was positive only when numerous spores were present, but the quality of the photomicrographs was superior after pretreatment with KOH. Pretreatment of sodium acetate-acetic acid-formalin-fixed fecal samples with 10% KOH and then a 5-min centrifugation time and staining with Weber modified trichrome stain provide for the excellent recovery of microsporidia in the routine diagnostic parasitology laboratory.     

A new trichrome-blue stain for detection of microsporidial species in urine, stool, and nasopharyngeal specimens.

Detection of microsporidia in clinical specimens has relied on electron microscopy, histology, or staining. This article describes further alterations to the modified trichrome staining method which make it easier to identify microsporidial spores. The changes are a decrease in the phosphotungstic acid level and the substitution of a colorfast counterstain, aniline blue, for the fast green of the original stain. The modified stain provides good contrast between microsporidial spores and background material including human and fungal cells. Stool specimens from 139 human immunodeficiency virus-seropositive patients revealed that 5 patients were infected with Enterocytozoon bieneusi and 6 patients had larger spores. Thin-section electron microscopy of the larger spores showed a structure consistent with that of either Encephalitozoon or Septata species. Three of the patients with Encephalitozoon- or Septata-like species had disseminated infection, with spores detected in nasopharyngeal aspirates and urine samples.     

Identification d’Enterocytozoon bieneusi par PCR dans les selles des patients immunodéprimés tunisiens

Introduction

Intestinal microsporidiosis is recognised as an important cause of opportunistic parasitosis in immunocompromised patients, especially HIV-infected patients. Enterocytozoon bieneusi is the common causal agent. The diagnosis of intestinal microsporidiosis has usually based on microscopic detection of the spores of microsporidia species in stool samples, requires additional staining techniques as Modified Weber's trichrome stain. However, the detection of the spores can be difficult and species determination, which is important for defining the appropriate treatment, is impossible. Polymerase chain reaction (PCR)-based methods have been successfully used for detection of microsporidian infections. They are more sensitive and are able to identify microsporidia species. The purpose of this study is to identify E. bieneusi to adapt treatment and assess the true prevalence of the intestinal microsporidiosis due to this species in compromised patients in Tunisia.

Patients and methods

One hundred and eighteen stools from immunocompromised patients, with a symptomatology in favour of the intestinal microsporidiosis, were analysed using light microscopy after staining with Modified Weber's trichrome stain and PCR.

Results

Only four were positive by Modified Weber's trichrome stain whereas eleven stools were positive by PCR, giving a prevalence of 20% in HIV-infected patients and 5,35% in human immunodeficiency virus-negative patients.

Conclusion

This study confirms the usefulness of PCR in the diagnosis of the intestinal microsporidiosis due to E. bieneusi. Indeed, PCR has greater sensitivity than Modified Weber's trichrome stain and can identify the species of microsporidia in order to adapt the treatment.     

Diagnosis of intestinal microsporidiosis by examination of stool and duodenal aspirate with Weber's modified trichrome and Uvitex 2B strains.

Severe, chronic diarrhea is a frequent complication of human immunodeficiency virus disease, and intestinal microsporidiosis is being recognized with increasing frequency in patients with AIDS. Noninvasive, cost-effective techniques are needed to optimize its diagnosis. Weber's modified trichrome stain (MTS) and the fluorochrome Uvitex 2B stain were used to detect microsporidial spores in smears of stool and duodenal aspirate (DA) samples received from human immunodeficiency virus-infected patients for examination for ova and parasites. Of 305 samples (292 stool and 13 DA samples) from 140 patients examined by MTS, 83 samples from 26 (18.6%) of the patients were positive for microsporidia (23 patients diagnosed initially and 3 diagnosed upon review). A subset of the samples studied by MTS consisting of 108 smears of stool and DA specimens from 60 patients was examined by Uvitex 2B. All 44 samples positive by MTS were also positive by Uvitex 2B. In addition, seven specimens and three patients were initially detected as positive by Uvitex 2B only (all three patients were positive also by MTS upon review). Confirmation of the diagnosis was obtained for 24 of 26 smear-positive patients by duodenal biopsy and/or stool transmission electron microscopy. Of 114 patients with stained smears negative for microsporidia, 23 had duodenal biopsies which showed no microsporidia. For the 43 patients who underwent duodenal biopsy, the sensitivity of both the MTS and the Uvitex 2B methods compared with biopsy results was 100%. Of six patients with negative duodenal biopsies and positive stained smears, four had microsporidia demonstrated by stool transmission electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. The Enteric Opportunistic Infections Working Group.

BACKGROUND. The diagnosis of infection with Enterocytozoon bieneusi, a microsporidian organism that causes chronic diarrhea in patients infected with the human immunodeficiency virus (HIV), has depended on invasive procedures. We have developed a new method to detect microsporidia spores in feces and duodenal aspirates. METHODS. Stool was obtained from four HIV-infected patients with biopsy-confirmed intestinal microsporidiosis. Slides prepared from unconcentrated, formalin-fixed stool specimens were stained with a new chromotrope-based technique and examined by light microscopy. Methods of stool concentration were also compared. The technique was then evaluated by examining 215 specimens from 134 HIV-infected persons with or without diarrhea. In addition, duodenal aspirates from 10 patients with unexplained chronic diarrhea were examined by light microscopy after staining according to the new and the traditional techniques. RESULTS. E. bieneusi spores were found in all unconcentrated stool specimens from the four patients with microsporidiosis. The use of various methods of stool concentration did not improve the detection of microsporidia spores. In the prospective study, microsporidiosis was detected in samples from 6 of 27 patients with chronic diarrhea, but in none of those from 42 patients with acute diarrhea or 65 patients without diarrhea. The presence of microsporidia spores in stool specimens and duodenal aspirates allowed the successful prediction of the presence of microsporidia in small-bowel biopsy specimens from all four patients who subsequently underwent endoscopy. CONCLUSIONS. E. bieneusi is an important cause of chronic diarrhea in HIV-infected persons. This new diagnostic technique serves as a practical, noninvasive means to detect microsporidia spores in stool specimens and is also applicable to the examination of duodenal aspirates.     

First detection of microsporidia in dairy calves in North America

Fecal specimens were obtained from a total of 413 dairy calves from farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. After removal of fecal debris by sieving and density gradient centrifugation, specimens were examined by fluorescence microscopy, polymerase chain reaction (PCR), and DNA sequencing analysis for the presence of microsporidia. Microscopic examination revealed no spores. PCR using generic primers for microsporidia revealed 70 positive calves. PCR was then conducted using specific primers for Enterocytozoon bieneusi, the most frequently found microsporidian in human infections. These primers revealed 13 positive calves from six farms in five states. DNA sequencing analysis of the 13 E. bieneusi-positive specimens confirmed the PCR results and indicated 96.8-99.8% similarity with E. bieneusi sequences in GenBank. This is the first report of E. bieneusi in cattle in North America.     

Zinc PVA versus potassium dichromate for preservation of microsporidian spores of human origin

Microsporidia are emerging opportunistic parasites. Preservation of the biological properties of microsporidian spores is often required in research work. The present study compared two preservatives; zinc polyvinyl alcohol (zinc PVA) and potassium dichromate solutions for preservation of microsporidian spores separated from human faecal samples. After 0, 1, 2 and 4 months of storage, morphological features and staining characters of the spores were assessed by light microscopy in modified trichrome-stained smears and their viability percentages were calculated using acridine orange/ethidium bromide mixture. Also, spore infectivity was evaluated by faecal spore shedding and intestinal spore load in mice orally inoculated with the preserved spores. Results revealed that morphological features, staining characters and viability of the spores were maintained in both solutions throughout the study period. Spore infectivity was completely preserved in zinc PVA solution but showed significant reduction in potassium dichromate solution at the fourth month of the preservation duration.     

Quantitative light microscopic detection of Enterocytozoon bieneusi in stool specimens: a longitudinal study of human immunodeficiency virus-infected microsporidiosis patients.

The clinical course of microsporidiosis caused by Enterocytozoon bieneusi and the pattern of intestinal shedding of spores have not been correlated, at least in part because detection of E. bieneusi in stools is more difficult than detection of other protozoa because of its smaller size and less intense staining. We examined with a modified trichrome stain 124 stool specimens collected over a 2-year follow-up period from 23 human immunodeficiency virus-infected patients with electron microscopic-proven E. bieneusi infection and correlated the results with electron microscopic observations from duodenal biopsy specimens taken at the beginning of the study period. E. bieneusi was detected in the stool at least once in 74% (17 of 23) of all patients, in 100% (9 of 9) of patients in whose tissue moderate or abundant numbers of parasites were seen, and in 57% (8 of 14) of patients in whose tissue few parasites were seen. In two patients with abundant tissue parasites, many microsporidia were detected in every stool specimen (13 of 13) during the follow-up period, whereas among the patients with few tissue parasites, only 23% (15 of 64) of stool specimens were positive. Furthermore, if spore stages as well as plasmodial stages were detected in tissue, stool specimens were more likely to be positive. Although most of the heavily infected stools were from patients with chronic diarrhea, microsporidia were detected in 33, 28, and 42% of stool specimens from patients with nil, intermittent, and chronic diarrhea patterns, respectively. Although quantitation of E. bieneusi spores in stool specimens was closely correlated with quantitation in tissue, it was not correlated with reported patterns of diarrhea.     

Comparative evaluation of modified trichrome and Uvitex 2B stains for detection of low numbers of microsporidial spores in stool specimens.

At present, the laboratory diagnosis of intestinal infections caused by microsporidia depends on the detection of the typical spores either with a modified trichrome stain (MTS) or by staining with fluorochromes. The purpose of the present study was (i) to compare staining with MTS (MTS method) and the staining with the fluorochrome Uvitex 2B (U2B method) with respect to their sensitivities and specificities, particularly in the presence of low numbers of spores, and (ii) to evaluate their reliabilities under routine laboratory conditions. First, 30 negative human stool specimens as well as 30 specimens enriched with a low concentration of microsporidial spores were examined. The U2B and MTS methods detected 27 and 30, of the positive samples, respectively (95% confidence intervals for sensitivity, 0.73 to 0.98 for the U2B method and 0.88 to 1.00 for the MTS method) without yielding false-positive results (95% confidence intervals for specificity, 0.88 to 1.00 for the MTS and U2B methods). In addition, analysis of serial dilutions of 17 stool specimens from AIDS patients containing microsporidia revealed comparable detection thresholds (P = 0.52) for both methods. Finally, 40 slides prepared from one stool specimen containing very few microsporidia and 40 negative slides were included in the routine diagnostic program during 1 month in order to monitor laboratory handling and run-to-run variations. Again, both methods exhibited comparable sensitivities (95% confidence intervals, 0.83 to 0.99 for the MTS method and 0.91 to 1.00 for the U2B method) and specificities (95% confidence intervals, 0.91 to 1.00 for the MTS and U2B methods). In conclusion, MTS and U2B methods are equally useful in the diagnosis of microsporidiosis. However, since detection thresholds for both methods differed considerably in all diluted stool specimens, performance of a combination of both methods may be more sensitive than the performance of only one procedure in the event of very low numbers of microsporidial spores.     

Identification of Encephalitozoon intestinalis in Travelers with Chronic Diarrhea by Specific PCR Amplification

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.     

Detection of <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis> in a new host—cockateel (<Emphasis Type="Italic">Nymphicus hollandicus</Emphasis>) using molecular methods

A total of 123 avian faecal specimens randomly collected in Bohemian commercial aviaries, Zoo parks and countryside were screened for the presence of human pathogenic microsporidia by both calcofluor M2R staining and polymerase chain reaction. Of these, no positive sample was detected using microscopical examination, and one isolate was detected by polymerase chain reaction and identified as Encephalitozoon cuniculi. Cockateel (Nymphicus hollandicus) represents a new avian host of this microsporidian.     

Detection by an Immunofluorescence Test of Encephalitozoon intestinalis Spores in Routinely Formalin-Fixed Stool Samples Stored at Room Temperature

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.     

A new acid-fast trichrome stain for simultaneous detection of Cryptosporidium parvum and microsporidial species in stool specimens.

The detection in stool specimens of Cryptosporidium parvum and microsporidia, the most frequent parasitic pathogens causing diarrhea in AIDS patients, until now has depended on two different staining methods. However, since double infections occur and minimization of laboratory costs is mandatory, development of a method for simultaneous detection of these parasites appeared desirable. We report on a new, inexpensive, and easy-to-perform staining procedure to demonstrate both acid-fast oocysts of C. parvum and other coccidia, as well as microsporidial spores. This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods and considerably reduces the time necessary for microscopic examination.     

Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray

Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microarray assay for the simultaneous detection and identification to the species level of four major microsporidian species: Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis. The 18S small-subunit rRNA gene was chosen as the amplification target, labeled with fluorescence dye, and hybridized to a series of species-specific oligonucleotide probes immobilized on a microchip. The specificity and sensitivity of the microarray were clearly demonstrated by the unique hybridization profiles exhibited by each species of microsporidian tested and its ability to detect as few as 10 spores. In order to assess the applicability of this microarray in a clinical setting, we conducted microarray assays of 20 fecal samples from AIDS patients. Twelve of these samples were positive for the presence of microsporidia and could be confidently identified; 11 of them were positive for more than one species. Our results suggested that this microarray-based approach represents an attractive diagnostic tool for high-throughput detection and identification of microsporidian species in clinical and epidemiological investigations.     

Detection of microsporidian spores in clinical samples by indirect fluorescent-antibody assay using whole-cell antisera to Encephalitozoon cuniculi and Encephalitozoon hellem.

Three polyclonal mouse antisera, to Encephalitozoon cuniculi, Nosema algerae, and Nosema corneum, and two polyclonal rabbit antisera, to E. cuniculi and Encephalitozoon hellem, were used in an indirect fluorescent-antibody assay (IFA) with Enterocytozoon bieneusi, E. cuniculi, and Encephalitgozoon. hellem spores (spores of the last two were taken from culture). Enterocytozoon bieneusi cannot be cultured. By IFA, antisera to E. cuniculi and E. hellem reacted strongly and equally with each other's spores. The mouse antisera reacted strongly with the homologous species, but for these there was segmental and particulate or "dot" staining of heterologous microsporidian spores, indicating cross-reactions with more selected antigens. In fecal samples, cross-reactions with both mouse and rabbit antisera were sometimes seen with different yeast species, with species of streptococci, and species of gram-negative rods. There were no cross-reactions to staphylococci. Enterocytozoon bieneusi was easily identified in duodenal and colonic biopsies, duodenal and colonic fluids, and feces of symptomatic AIDS patients by IFA. In a study of 12 AIDS patients with diarrhea, the new IFA identified microsporidia in all of 11 fecal samples, three colon fluids, six duodenal fluids, and three duodenal biopsy touch preparations. Although the fecal sample of 1 of the 12 was negative, the patient's duodenal fluid contained microsporidian spores by IFA.     

Detection of microsporidia (Enterocytozoon bieneusi) in intestinal biopsy specimens from human immunodeficiency virus-infected patients by PCR.

Intestinal microsporidiosis has been implicated as a major cause of chronic diarrhea in human immunodeficiency virus (HIV)-infected patients. So far diagnosis depends on direct visualization of the parasites by light and transmission electron microscopy. We evaluated the diagnostic value of microsporidian DNA amplification by PCR on duodenal biopsy specimens obtained from patients with and without intestinal microsporidiosis caused by Enterocytozoon bieneusi. Thirteen HIV-infected patients (all CDC stage C3) were studied. Eight patients had intestinal microsporidiosis caused by E. bieneusi (n = 6), Septata intestinalis (n = 1), and Encephalitozoon cuniculi (n = 1); microsporidioses were diagnosed by light microscopy of stool samples and confirmed by light and electron microscopy of intestinal biopsy specimens. Five patients had no microsporidia in their stool samples or in their intestinal biopsy specimens, as examined by light and electron microscopy. Additionally, DNA prepared from Toxoplasma gondii derived from mouse ascites was used as a further control. A 353-bp DNA fragment of the small-subunit rRNA gene could be amplified from all six biopsy specimens infected with E. bieneusi, and the nature of the PCR products was confirmed by Southern blot hybridization. No amplification of DNA fragments was seen by using DNA extracted from biopsy specimens with S. intestinalis or E. cuniculi infection or without microsporidian infection and with template DNA extracted from T. gondii. The results suggest that PCR testing of intestinal biopsy specimens may be a useful approach to diagnosing microsporidiosis in HIV-infected patients.     

Diagnosis of intestinal microsporidiosis in pediatric oncology patients in Egypt using modified acid fast trichrome staining versus PCR

Microsporidia are intracellular opportunistic parasites that cause chronic diarrhea in AIDS patients. Little is known about the prevalence of these pathogens in pediatric cases with cancer and diarrhea. Unidentified causes of chronic diarrhea were previously encountered in pediatric cancer patients at the National Cancer Institute in Egypt. Therefore, this study tried to search for the contribution of microsporidia as a causative agent of diarrhea in this population using acid-fast trichrome stain as a specific staining and the PCR in order to evaluate the staining technique in clinical diagnosis of microsporidia. Between January 2008 and June 2009, 271 diarrheic samples from pediatric patients with cancer were studied. Microsporidia were confirmed in 13 (4.8%) cases by both PCR and staining, and additional 2 samples were positive only by staining. As a negative control, stool samples from 60 diarrheic children without malignant cancer and no microsporidia detection were examined by these two methods. So, it can be concluded that adding a diagnostic test for microsporidia to the clinical laboratory work in hospitals concerned with cancer is essential. Acid fast trichrome staining technique as being nearly efficacious as the PCR, but simpler and less expensive, can replace the molecular techniques for the diagnosis of microsporidia.     

A Case of Enterocytozoon bieneusi Infection in an HIV-Negative Renal Transplant Recipient

 Reported here is a case of microsporidiosis that occurred in an HIV-negative renal transplant recipient. The patient developed protracted diarrhea 18 months following transplant surgery. Many spores of Enterocytozoon bieneusi were detected in stool smears using a modified trichrome staining method. Identification was confirmed using the polymerase chain reaction. Histological examination of duodenal biopsies revealed numerous spores in the cytoplasm of enterocytes. Tacrolimus and steroid regimens were decreased, treatment with mycophenolate mofetil was discontinued, and the patient was given albendazole and metronidazole for 2 weeks. The diarrhea resolved after 15 days of treatment; 2 months later the patient had recovered completely. A more systematic search for microsporidia using specific staining procedures should be performed in transplant recipients who develop severe diarrhea.