Since the first description in 1985 , the microsporidian parasite Enterocytozoon bieneusi and more recently Encephalitozoon intestinalis (previously named Septata intestinalis)  have been frequently detected in small intestinal enterocytes of patients infected with HIV. The high prevalence of microsporidiosis in up to 50% of HIV-infected patients with chronic diarrhoea [3–9] provides strong circumstantial evidence for an enteropathogenic role of microsporidia. However, a recent study reported the occurrence of microsporidia in about 30% of HIV-infected patients both with and without diarrhoea . In addition, the mechanism by which microsporidia might cause diarrhoea is unclear. Histological alterations of infected epithelial cells range from only minor changes [6,9,11] to severe cell injury [12,13], and only limited data exist about abnormalities of the intestinal mucosa in HIV-infected patients with microsporidia [14,15]. In our ongoing studies of gastrointestinal manifestations of HIV infection, we therefore prospectively investigated the prevalence of microsporidiosis in HIV-infected patients with and without diarrhoea. In addition, we compared mucosal architecture and brush border enzyme activities in patients with microsporidiosis, patients with enteropathogens other than microsporidia, and patients without detectable enteropathogens.
Materials and methods
Two-hundred and fifty-nine consecutive patients undergoing diagnostic oesophagogastroduodenoscopy were enrolled in this prospective study. Endoscopy was performed because of dysphagia, nausea, vomiting, epigastric and abdominal pain, diarrhoea, unexplained weight loss or fever of unknown origin. Patients were grouped by the presence or absence of acute or chronic diarrhoea, defined as more than three loose bowel movements per day for at least 1 week or 1 month, respectively. All patients gave written informed consent and the study was approved by the local ethics committee.
All patients had a complete physical examination and their medical history was recorded using a standardized protocol. All patients underwent oesophagogastroduodenoscopy and biopsies were taken from the distal duodenum. Biopsies and at least two stool samples were examined for the presence of viral, bacterial or protozoal pathogens as previously described . In brief, biopsies were examined by immunohistology for the presence of cytomegalovirus (CMV) or herpesvirus infections, and electron microscopy was performed to detect microsporidia. Stools were cultured for enteropathogenic bacteria, including mycobacteria, and screened for the presence of Clostridium difficile toxin. Furthermore, stool samples were stained according to the modified Kinyoun protocol and the modified trichrome technique to detect cryptosporidia and microsporidia, respectively. In addition, stool samples were screened by electron microscopy for the presence of stool virus. If possible, within 4 weeks after establishment of the diagnosis, absorptive function was tested in patients harbouring microsporidia by a breath hydrogen (H2) test after administration of 50 g lactose, a blood D-xylose test, and a Schilling test after intrinsic factor replacement.
Brush border enzyme activities were measured in 19 patients with microsporidia, 14 HIV-infected patients with enteropathogens other than microsporidia, and 19 HIV-infected patients without enteropathogens. The three groups did not differ in age, sex, risk behaviour or stage of disease (each P > 0.05). Patients with microsporidia and those with enteropathogens other than microsporidia did not differ in the number of CD4 T lymphocytes (P > 0.05). However, patients with microsporidia had lower CD4 T lymphocytes than patients without enteropathogens (median, 8 versus 57 × 106/l; P = 0.02).
Activities of lactase, α-glucosidase and alkaline phosphatase were measured at the Benjamin Franklin University Clinic as described by Gutschmidt et al. [17,18]. Enzyme activity was measured by incubating unfixed 10 µm cryostat sections of snap-frozen distal duodenal biopsy specimens at 37°C in a moist chamber. For the detection of lactase activity, 2.24 mmol/l of 5-Br-4-Cl-3-indoxyl-β-D-fucoside (Bachem, Bubendorf, Switzerland) as substrate in citrate-phosphate buffer (pH 6) with an Fe2+/Fe3+ redox system as additional reagent for the development of the indigo dye deposits in the brush border membrane was applied for 20 min. For α-glucosidase, 10 mmol/l 2-napthol-α-D-glucoside (Sigma, Deisenhofen, Germany) was used as substrate in hexazonium-p-rosanilin as coupling reagent, applied for 10 min. For alkaline phosphatase, 1.5 mmol/l naphtol-AS-BI-phosphate (Sigma) was used as substrate in Tris-HCl buffer (pH 8.3) with Fast Blue B (Serva, Heidelberg, Germany) as coupling reagent, applied for 5 min. Absorbance measurement was performed on a Leitz MPV2 microdensitometer (Leitz, Wetzlar, Germany) at 640 nm for lactase, 550 nm for alkaline phosphatase, and at 480 nm for α-glucosidase, using fields of 4 × 4 µm in the brush border region. The transition zone between the lower and medium third of the villus was defined as basal measuring point, and the transition zone between the medium and the apical third of the villus was defined as apical measuring point. Uncoloured tissue served as blank (100% transmission). Ten measurements per measuring position were averaged. Based on a variance component analysis, we found a measurement variability (SD) for apical alkaline phosphatase of 0.261, for basal alkaline phosphatase of 0.228, for apical α-glucosidase of 0.175, for basal α-glucosidase of 0.182, for apical lactase of 0.202, and for basal lactase of 0.092.
Villus height, villus surface area, crypt depth, and the number of mitotic figures per crypt were measured in duodenal biopsies from 16 patients with microsporidia, 21 HIV-infected patients with enteropathogens other than microsporidia, and 29 HIV-infected patients without enteropathogens. The three groups did not differ in age, sex, risk behaviour, or stage of disease (each P > 0.05). Patients with microsporidia and those with enteropathogens other than microsporidia did not differ in the number of CD4 T lymphocytes (P > 0.05). However, patients with microsporidia had lower CD4 T lymphocytes than those without enteropathogens (median, 12 versus 46 × 106/l; P = 0.007).
Morphometry of the duodenal mucosa was performed at the Benjamin Franklin University Clinic as described by Clarke . Briefly, distal duodenal biopsy specimens were fixed in ethanol-glacial acetic acid (3 : 1, vol/vol) for 24 h and then rinsed with and stored in 75% ethanol. They were then stained using the Feulgen reaction before the individual villi were liberated by microdissection under a stereomicroscope. From each sample, 10 random isolated villi were chosen for the determination of their height, width, and breadth both at the base and the apex. From these values the surface area of an individual villus was calculated. Subsequently, the individual crypts were dissected and their lengths recorded. The total number of mitotic figures per crypt was counted. The means of the morphometric data for the individual specimen were compared.
Measurement results were described as medians and range between 25th and 75th percentile. More than two independent samples were first compared using the Kruskal-Wallis test (non-parametric analysis of variance); if significant differences were found, two groups were compared by the two-tailed Mann–Whitney U test. The frequency of abnormalities in different groups of patients was compared using the χ2 test. P values <0.05 were considered significant.
Prevalence of microsporidiosis
Microsporidia were detected by electron microscopy in duodenal biopsies from 19 patients (7.3%; 19 out of 259). Risk factors in all 19 patients were male-to-male sexual contacts. In 17 of these patients E. bieneusi was detected in enterocytes, and in two patients E. intestinalis was detected in enterocytes and macrophages of the lamina propria. The prevalence of microsporidia in patients with diarrhoea was 13.8% (17 out of 123), in the subgroup of patients with chronic diarrhoea the prevalence was even higher (19.1%; 13 out of 68; Table 1). Detection of microsporidia was associated with diarrhoea (P < 0.0001), although two patients with detectable microsporidia had no diarrhoea. In one of these patients without diarrhoea E. intestinalis was detected, and in the other patient E. bieneusi was found. The patient infected with E. bieneusi and without diarrhoea had a CD4 count of 164 × 106/l. All other patients with microsporidia had CD4 T lymphocytes <80 × 106/l. Diarrhoea was more common in patients with microsporidia than in patients without enteropathogens and CD4 T lymphocytes < 200 × 106/l [89.5% (17 out of 19) versus 31% (39 out of 122); P < 0.0001].
Because the lower CD4 counts in patients with chronic diarrhoea compared with patients who did not have diarrhoea could indicate that diarrhoea was related to advanced immunodeficiency rather than micro-sporidiosis, additional analyses were performed in the subgroup of patients with CD4 T cells < 50 × 106/l. In the absence of detectable enteric infections, patients with CD4 counts of <15, 15–29, or 30–49 × 106/l had a similar prevalence of acute [five out of 35 (14%), seven out of 24 (29%), and four out of 25 (16%), respectively] or chronic [six out of 35 (17%), one out of 24 (4%), and four out of 25 (16%), respectively] diarrhoea (P = 0.42). Thus, when CD4 T cells fell to <50 × 106/l, further progression of immunodeficiency was not associated with an increased prevalence of diarrhoea. Furthermore, in patients with CD4 T cells <50 × 106/l, microsporidia were more common in patients who had acute [four out of 37 (11%)] and chronic [10 out of 50 (20%)] diarrhoea than in patients who had no diarrhoea [one out of 79 (1.3%); P = 0.0013]; the same was true for patients with CD4 T cells < 15 × 106/l [two out of 16 (13%) and five out of 27 (19%) versus none out of 37 patients with microsporidia, respectively; P = 0.029].
Co-infections with other enteropathogens were found in 58% of patients with microsporidiosis (11 out of 19). Seven out of eleven patients (64%) with microsporidiosis had co-infections with CMV. Of these seven CMV-infected patients, two patients had additional infections with cryptosporidia, one with mycobacteria, one with C. difficile, and one with both mycobacteria and C. difficile. Enterovirus and Entamoeba histolytica were each found as coinfecting agents in two patients with microsporidia. Two other patients had co-infections with adenovirus, one of these patients with additional coronavirus, the other together with Giardia lamblia. There was no difference in the frequency of co-infections between patients with microsporidiosis and patients without microsporidia and a CD4 count < 200 × 106cells/l [58% (11 out of 19) versus 42% (90 out of 212); P = 0.085]. Additional enteropathogens did not influence the duration [3 months (range, 2–18) versus 7.5 months (range, 2–12) in microsporidia-infected patients with and without additional enteropathogens, respectively; P > 0.05] and frequency of diarrhoea [seven (range, 5–10) versus eight (range, 5–10) loose bowel movements per day in microsporidia-infected patients with and without additional enteropathogens, respectively; P > 0.05]. Furthermore, in the subgroup of patients with CD4 T cells < 50 × 106/l, diarrhoea was more common in patients exclusively harbouring microsporidia than in patients without enteropathogens (five out of five versus 27 out of 84; P = 0.009).
Of the patients with microsporidia, 75% (nine out of 12) had a lactase deficiency measured by the H2 exhalation test. In 83% (10 out of 12) of the patients with microsporidia, a diminished D-xylose absorption was detected, as measured by blood D-xylose test, and in 88% (seven out of eight) of patients, abnormal vitamin B12 absorption was found, as measured by the Schilling test after intrinsic factor replacement. There was no relationship between the duration of diarrhoea or the frequency of loose bowel movements per day and the degree of the functional abnormality (each P < 0.05).
Brush border enzymes
Patients with microsporidia more frequently had lactase deficiency than HIV-infected patients with enteropathogens other than microsporidia (P = 0.0038) or patients without enteropathogens (P = 0.03; Table 2). Absence of brush border lactase correlated with abnormal H2 exhalation in all patients tested. Comparing patients with detectable lactase, enzyme activities were not different between the three groups at either measuring point. Patients with microsporidia had a significantly decreased activity of alkaline phosphatase (P = 0.028) and α-glucosidase (P = 0.025) at the basal but not at the apical measuring point compared with patients without enteropathogens. In patients with microsporidiosis, enzyme activities were not different between patients who did or did not have additional enteropathogens (P > 0.05). Of note, both patients with microsporidiosis but no diarrhoea had detectable lactase, and the highest activity of lactase was found in the patient infected with E. bieneusi who had a CD4 count > 100 × 106/l who did not have diarrhoea. In the total group of patients investigated, patients with diarrhoea more frequently had lactase deficiency than patients without diarrhoea (P = 0.039). The activities of alkaline phosphatase and α-glucosidase were not different in patients with and without diarrhoea (P > 0.05). There was no correlation between enzyme activities and the number of CD4 T lymphocytes in the peripheral blood.
Patients with microsporidia had reduced villus height and reduced villus surface area compared with patients with enteropathogens other than microsporidia (P = 0.0154 for villus height, P = 0.0084 for villus surface; Table 3) and compared with patients without enteropathogens (P = 0.043 for villus height, P = 0.0041 for villus surface). Villus surface area was reduced by about 40% in patients with microsporidia compared with HIV-infected patients with enteropathogens other than microsporidia and without enteropathogens. Crypt depth was significantly increased in patients with microsporidia compared with patients without enteropathogens (P = 0.0032). These parameters were not different between patients with microsporidia who had or had no additional infections (P > 0.05). Patients with or without diarrhoea did not differ in villus height and villus surface area. Patients with enteropathogens other than microsporidia and patients without enteropathogens did not differ significantly in mucosal architecture. Patients with absent lactase activity had lower villus surface area than patients with detectable lactase activity [0.130 mm2 (range, 0.890–0.166 mm2) versus 0.251 mm2 (range, 0.236–0.268 mm2); P = 0.009]. Villus height and villus surface area did not correlate with activities of alkaline phosphatase and α-glucosidase.
Microsporidia are one of the most frequent agents detected in AIDS patients with diarrhoea [4,8,9,12–14,20], but their pathogenic relevance was recently questioned by the detection of microsporidia in similar proportions of HIV-infected patients with and without diarrhoea . In our study of 123 patients with diarrhoea and 136 patients without diarrhoea, microsporidiosis was found to be significantly associated with diarrhoea. This correlation was also found in patients stratified for very low CD4 counts, and in the absence of secondary enteropathogens, a decrease of CD4 T cells < 50 × 106/l was not associated with an increased prevalence of diarrhoea. These findings strongly suggest that microsporidia, but not immunodeficiency per se, cause diarrhoea in AIDS patients. The only patient who had E. bieneusi infection but no diarrhoea had a CD4 T-cell count almost threefold higher than the other patients with microsporidia. This could indicate that the severity and clinical course of microsporidiosis varies with immune status as described for cryptosporidiosis . Co-infections with other enteropathogens were found in previous studies in up to 37% of patients with microsporidiosis [4,8,20], and the rate of co-infections in our study cohort was even higher. This could be due to our extensive evaluation of duodenal biopsies and at least two stool samples per patient for the presence of enteropathogens. However, frequency and duration of diarrhoea in patients with microsporidiosis was independent of the presence of additional enteropathogens. Therefore, co-infections do not explain the correlation between microsporidiosis and diarrhoea. With a prevalence of 19.1% in patients with chronic diarrhoea, microsporidia are therefore the second most common enteropathogens after CMV in Berlin AIDS patients.
Diarrhoea in HIV-infected patients with microsporidiosis could in part be due to malabsorption [8,9,15,22]. All patients with microsporidiosis had reduced absorptive function as demonstrated by malabsorption of D-xylose, vitamin B12, and lactase each in about 80%. Since lactose is absorbed in the proximal small intestine and vitamin B12 in the ileum, these findings indicate a reduced absorptive function throughout the small intestine in patients with microsporidiosis. In accordance with clinical tests of H2 exhalation, lactase deficiency was also found by in situ measurements of brush border enzymes in the duodenal mucosa. Lactase deficiency was significantly more frequent in patients with microsporidia, even when compared with patients harbouring enteropathogens other than microsporidia. Similarly, in patients with microsporidiosis, villus height and villus surface area were significantly lower than in patients without enteropathogens or in patients with enteropathogens other than microsporidia. The reduction of absorptive surface by about 40% compared with patients with enteropathogens other than microsporidia indicates an especially damaging effect of microsporidiosis. Increased cell sloughing in patients with microsporidia is the most likely explanation for the reduction of villus surface area, since crypt depth was increased and mitotic activity was at least as high as in patients with other or no enteropathogens, which argues against hyporegeneration. Apical enterocyte desquamation could furthermore explain the high frequency of lactase deficiency found in our study, because lactase is expressed predominantly on cells at the upper third of the villus.
In accordance with a report from Greenson et al. , villus and crypt alterations did not correlate with the presence of diarrhoea. However, lactase deficiency was significantly associated with diarrhoea, and patients with absent lactase activity had lower villus surface area than patients with detectable lactase activity. Thus, patients with microsporidia might profit from dietary exclusion of lactose. Activities of alkaline phosphatase and α-glucosidase did not correlate with either mucosal architecture or diarrhoea. Surprisingly, in contrast to the predominant localization of the parasites at the villus tips, patients with microsporidiosis showed a reduction of activities of alkaline phosphatase and α-glucosidase not at the apical, but at the basal part of the villus. These findings could possibly indicate an indirect mechanism of enterocyte damage in addition to the direct effect of microsporidial infection of enterocytes. In accordance with literature , no inflammatory cell infiltrate was detectable in our patients with microsporidia. However, an increase in epithelial lymphocytes has been described in patients with microsporidiosis , which could be involved in such an indirect mechanism of cell damage. A loss of cells throughout the villus could explain the reduction in alkaline phosphatase and α-glucosidase found in the basal part of the villus. Although we and others  found no histological evidence for this hypothesis, our study does not rule out this possibility.
In summary, our study confirms the association between microsporidia and diarrhoea. The pathophysiologic mechanism by which microsporidia cause diarrhoea appears in part to be malabsorptive, caused by a reduction of absorptive mucosal surface area and enterocyte immaturity with impairment of function.
The authors are indebted to G. Bogusch, C. Brunn, U. Dethlefs, M. Lemke, B. Rieger, S. Münchow and U. Schreiber for expert technical assistance and to U. Littke and W. Kronitz for data documentation.
1. Desportes I, Charpentier YL, Galian A: Occurrence of a new microsporidian: Enterocytozoon bieneusi n.g., n.sp., in the enterocytes of a human patient with AIDS
. J Protozoal
2. Cali A, Kotler DP, Orenstein JM: Septata intestinalis n.g., n.sp., an intestinal microsporidian associated with chronic diarrhea and dissemination in AIDS patients
. J Euk Microbiol
3. Lucas SB, Papadaki L, Conlon C, Sewankambo N, Goodgame R, Serwadda D: Diagnosis of intestinal microsporidiosis in patients with AIDS
. J Clin Pathol
4. van Gool T, Snijders F, Reiss P, et al.
: Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV by a new rapid fluorescence technique
. J Clin Pathol
5. Greenson JK, Belitsos PC, Yardley JH, Bartlett JG: AIDS enteropathy: occult enteric infections and duodenal mucosal alterations in chronic diarrhea
. Ann Intern Med
6. Eeftinck Schattenkerk JK, van Gool T, van Ketel RJ, et al.
: Clinical significance of small-intestinal microsporidiosis in HIV-1-infected individuals
7. Field AS, Hing MC, Milliken ST, Marriott DJ: Microsporidia in the small intestine of HIV-infected patients. A new diagnostic technique and a new species
. Med J Aust
8. Kotler DP, Orenstein JM: Prevalence of intestinal microsporidiosis in HIV-infected individuals referred for gastroenterological evaluation
. Am J Gastroenterol
9. Molina J, Sarfat C, Beauvais B, et al.
: Intestinal microsporidiosis in human immunodeficiency virus-infected patients with chronic unexplained diarrhea: prevalence and clinical and biologic features
. J Infect Dis
10. Rabeneck L, Gyorkey F, Genta RM, Gyorkey P, Foote LW, Risser JMH: The role of microsporidia in the pathogenesis of HIV-related chronic diarrhea
. Ann Intern Med
11. Modigliani R, Bories C, Charpentier YL, et al.
: Diarrhoea and malabsorption in acquired immune deficiency syndrome: a study of four cases with special emphasis on opportunistic protozoan infestations
12. Orenstein JM, Chiang J, Steinberg W, Smith PD, Rotterdam H, Kotler DP: Intestinal microsporidiosis as a cause of diarrhea in human immunodeficiency virus-infected patients. A report of 20 cases
. Hum Pathol
13. Peacock CS, Blanshard C, Tovey DG, Ellis DS, Gazzard BG: Histological diagnosis of intestinal microsporidiosis in patients with AIDS
. J Clin Pathol
14. Kotler DP, Francisco A, Clayton F, Scholes JV, Orenstein JM: Small intestinal injury and parasitic diseases in AIDS
. Ann Intern Med
15. Kotler DP, Reka S, Chow K, Orenstein JM: Effects of enteric parasitoses and HIV infection upon small intestinal structure and function in patients with AIDS
. J Clin Gastroenterol
16. Schmidt W, Schneider T, Heise W, et al.
: Stool virus, coinfections, and diarrhea in HIV-infected patients
. J Acquir Immune Defic Syndr
17. Gutschmidt S, Lange U, Riecken EO: Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum. A quantitative histochemical study
18. Gutschmidt S: ‘In situ’determination of apparent Km and Vmax of brush border disaccharidases along the villi of normal human jejunal biopsy specimens. A quantitative histochemical study
19. Clarke RM: Mucosal architecture and epithelial cell production rate in the small intestine of the albino rat
. J Anat
20. Weber R, Bryan RT, Owen RL, et al.
: Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates
. N Engl J Med
21. McGowan I, Hawkins AS, Weller IVD: The natural history of cryptosporidial diarrhoea in HIV-infected patients
22. Lambl BB, Federman M, Pleskow D, Wanke CA: Malabsorption and wasting in AIDS patients with microsporidia and pathogen-negative diarrhea
23. Weber R, Bryan RT, Schwartz DA, Owen RL: Human microsporidial infections
. Clin Microbiol Rev