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Background Cholangitis in patients with the acquired immunodeficiency syndrome (AIDS) is usually associated with opportunistic infections by cryptosporidium species or cytomegalovirus, but in about a third of cases no opportunistic agent is identified. We suspected some of these cases of biliary disease might be explained by infection with the microsporidia species Enterocytozoon bieneusi, an obligate intracellular protozoan that causes chronic diarrhea in patients infected with the human immunodeficiency virus (HIV).
Methods We studied eight HIV-infected homosexual men (in either group IV of the classification of the Centers for Disease Control and Prevention or group II, with a CD4 cell count of
Results All eight patients with unexplained AIDS-related cholangitis had biliary microsporidiosis. Intraepithelial E. bieneusi spores (1 to 2 microm) and supranuclear plasmodia (3 to 8 microm) were identified in the six duodenal-biopsy specimens. May-Grunwald-Giemsa staining of bile samples revealed free forms of microsporidia in all eight patients, and the presence of E. bieneusi was confirmed by electron microscopy. E. bieneusi was also identified in ductal biliary cells on a liver biopsy, in one common-bile-duct smear, and in gallbladder epithelium (in two patients). Four patients were found to have associated but previously undetected biliary or duodenal cryptosporidiosis, whereas another had biliary infection associated with cytomegalovirus.
Conclusions Infection of the biliary tract with E. bieneusi is associated with and may be a cause of AIDS-related cholangitis.
10 per cubic millimeter) who were referred because of cholangitis for which no causative agent had been found by standard tests. All the patients underwent abdominal ultrasonography and endoscopic ultrasonography or endoscopic retrograde cholangiopancreatography with collection of bile from the common bile duct. One patient had transhepatic biliary catheterization, and two others had cholecystectomy. Bile samples, duodenal- and liver-biopsy specimens, and gallbladder tissue were studied by light and electron microscopy.
No opportunistic agent is identified in about 40 percent of patients with AIDS-related biliary disease. It has been suggested that the immunodeficiency itself, human immunodeficiency virus (HIV) infection of the bile-duct mucosa, and other, unidentified pathogens may cause AIDS-related cholangitis. Several syndromes have been attributed to microsporidia infections in AIDS5,6,7,8. Two cases of biliary tract infection by Enterocytozoon bieneusi have now been described,9,10 suggesting that microsporidiosis could be involved in AIDS-related cholangitis. We therefore conducted a prospective study in an attempt to identify microbial causes of unexplained AIDS-related biliary vesicular disease, with special attention to microsporidia infections.
Methods
Patients
Over an 18-month period (April 1990 through September 1991), eight HIV-positive homosexual men were referred to our liver unit with unexplained chronic cholestasis or diarrhea. Their mean (±SD) age was 38 ±6 years, and HIV infection had been diagnosed a mean of 3.6 ±1.3 years previously.
Six patients met the criteria for AIDS established by the Centers for Disease Control and Prevention (CDCP)11 (group IV-C1 or IV-D); the remaining two were asymptomatic (group II) but had low CD4 lymphocyte counts (8 and 10 per cubic millimeter). Six of the eight patients had p24 antigenemia (Table 1). In six of the eight patients opportunistic infections and neoplasms preceded the onset of cholangitis or were present when it was diagnosed; they included cytomegalovirus chorioretinitis (three patients), Pneumocystis carinii pneumonia (two), cytomegalovirus viremia (one), oral candidiasis (one), meningeal cryptococcal infection (one), and Kaposi's sarcoma (two). All but one of the patients had chronic diarrhea or cholestasis (serum alkaline phosphatase and 
-glutamyltransferase activities at least 1.5 times the upper limit of normal). None had a history of biliary tract surgery. All the patients had abdominal pain (in the epigastric region in one, in the right upper quadrant in five, and in both locations in two). None had jaundice, five had persistent fever, and none had acute cholangitis before undergoing endoscopic retrograde cholangiopancreatography (ERCP) or endoscopic ultrasonography. Six of the eight patients had marked weight loss (i.e., a loss of more than 10 percent of their normal weight) (Table 2). Serum alkaline phosphatase and -glutamyltransferase activities were elevated in most patients, with mean elevations 2.3 times the upper limit of normal (range, 0.6 to 7.6) and 2.7 times the upper limit of normal (range, 0.8 to 7.0), respectively. Serum aminotransferase activities were elevated in seven patients, with means of 2.9 and 2.1 times the upper limit of normal for aspartate aminotransferase and alanine aminotransferase, respectively (ranges, 0.7 to 13.2 and 0.5 to 8.4). The serum total bilirubin concentrations were below 1.0 mg per deciliter (17 µmol per liter) in all the patients (mean, 0.6 mg per deciliter [9.6 µmol per liter]) (Table 2).
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It is noteworthy that these eight patients had no epidemiologic relation to each other and were not clustered with respect to time or location. They had no exposure that was related to recent travel overseas.
Morphologic Procedures
All the patients underwent abdominal ultrasonography, and seven underwent endoscopic ultrasonography with the collection of a biliary sample by cannulation of the common bile duct. Four patients underwent ERCP, including endoscopic sphincterotomy in two patients. The morphologic diagnosis of cholangitis was based on Cello's criteria3. Two patients (Patients 7 and 8) underwent surgical cholecystectomy for acalculous cholecystitis associated with cholangitis. Endoscopy of the upper gastrointestinal tract (with duodenal biopsy) and colonoscopy (with staged colonic biopsies) were performed in seven and five patients, respectively.
Microbiologic Procedures
All bile and tissue samples were processed for microbiologic tests as described below. Bile samples were obtained after endoscopic papillary cannulation in seven patients. Further bile samples were obtained from Patient 3 by means of transhepatic catheterization and from Patient 8 during surgical cholecystectomy; a bile sample was obtained during cholecystectomy from the patient who did not undergo endoscopic ultrasonography (with this procedure, duodenal contamination is avoided). Papillary or choledochal biopsies were performed in four patients during ERCP, and transparietal liver-biopsy specimens were obtained from two patients. Biopsy specimens from the duodenum and liver, gallbladder tissue, and bile samples were studied by light and electron microscopy.
Microscopy
Light Microscopy
Touch preparations of the tissue samples and cytocentrifugation smears of bile were dried, stained with May-Grunwald-Giemsa for 30 minutes, fixed in Bouin's fixative for at least 4 hours, and then processed routinely. Slides of each specimen were stained with hematoxylin and eosin, May-Grunwald-Giemsa, Gram's, Ziehl-Neelsen, and methenamine-silver stains. Glutaraldehyde-fixed material for electron microscopy was also embedded in paraffin. Some sections were stained with toluidine blue and examined by light microscopy.
Electron Microscopy
Bile samples were centrifuged, and the pellets containing microsporidia spores were fixed in 2 percent glutaraldehyde in 1 percent 0.1 M sodium cacodylate buffer for two hours, rinsed in buffer, and postfixed in a medium containing 1 percent (vol/vol) osmium tetroxide and potassium ferricyanide in cacodylate buffer for one hour at room temperature. After ethanol dehydration, the material was embedded in Spurr's resin. Ultrathin sections were stained with uranyl acetate and lead citrate. They were observed with a Jeol transmission electron microscope (JEM 100CX) at 80 kV.
Results
Six of the eight patients with unexplained chronic diarrhea or cholestasis had AIDS-related cholangitis at the time of their first admission, as shown by abdominal ultrasonography, endoscopic ultrasonography, or ERCP. The morphologic abnormalities included dilatation of both the intrahepatic and the common bile ducts, irregularities of the bile-duct wall, and an abnormal gallbladder (with wall thickening, distention, or sludge). Papillary stenosis was also present in one patient, and hyperalgesic cholecystitis associated with intrahepatic and extrahepatic abnormalities necessitated cholecystectomy in two patients. One patient (Patient 2), with a seven-month history of unexplained diarrhea, had a normal ERCP; four months later, a second ERCP confirmed the diagnosis of cholangitis. Another patient (Patient 6) had a normal biliary tract on repeated ultrasonography and endoscopic ultrasonography and remained free of biliary disease after 12 months of follow-up, despite the onset of AIDS. Finally, a typical AIDS-related cholangitis appeared 15 months after the diagnosis of biliary infection with microsporidia. Despite normal morphologic findings, this infection was diagnosed in both these patients at the time of the first ERCP (see below).
Standard endoscopic exploration of the upper digestive tract and colon was either normal or showed nonspecific abnormalities in the seven and five patients who underwent the respective procedures.
The results of microbiologic tests using light and electron microscopy are summarized in Table 3. Intraepithelial E. bieneusi spores (1 to 2 microm) and supranuclear plasmodia (3 to 8 microm) were found in duodenal-biopsy specimens (with hematoxylin-eosin and May-Grunwald-Giemsa staining) from six patients. Bile cultures were negative in all but one patient, in whose case they yielded Klebsiella pneumoniae (in a bile specimen obtained by ERCP). May-Grunwald-Giemsa staining revealed free forms of microsporidia in the bile of all eight patients. Toluidine-blue-stained slides and electron microscopy (when performed) of the duodenal-biopsy specimen and bile confirmed the presence of E. bieneusi (Figure 1A and Figure 1B). Intracellular plasmodia were identified in common-bile-duct smears from one patient and in samples of gallbladder epithelium from two patients. Intraepithelial plasmodia and spores of E. bieneusi were found in ductal biliary cells in the liver-biopsy specimen from one patient (Figure 2).
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Three patients received metronidazole treatment (1 g twice daily for 15 days), and one was given quinacrine (125 g twice daily for 15 days), with no effect on diarrhea, cholestasis, or biliary microsporidia.
Discussion
Infection of the bile ducts with microsporidia appears to be far from rare. It was found in all eight patients with AIDS-related cholestasis whom we observed over an 18-month period and who were free of the usual pathogens encountered in this setting, namely cryptosporidium and cytomegalovirus.
Infections due to microsporidia are reported increasingly in immunosuppressed patients, and as a result pathologists are more aware of this organism5,6,7,8,9,10,12,13,14,15,16. The diagnosis is based on the examination of smears with May-Grunwald-Giemsa staining and tissue sections stained with hematoxylin and eosin, together with electron microscopy to distinguish species. Our results confirm that light microscopy is efficient in screening for microsporidia-related biliary infections in HIV infection12,13,14,15. The optimal stain for light microscopy was toluidine blue on resin-embedded material; this stain gives good contrast between the spores and the background and permits histologic diagnosis at lower magnification.
Biliary microsporidiosis is associated with microsporidiosis of the small intestine in persons infected with HIV type 1, and it usually results in AIDS-related cholangiopathy, including cholangitis or acalculous cholecystitis. However, cholangitis developed in one of our eight patients with E. bieneusi infection of the bile after 15 months of follow-up. Biliary microsporidiosis might be thought to result from duodenal contamination during retrograde endoscopic procedures, but the identification of E. bieneusi in biliary cells obtained by liver biopsy, transhepatic catheterization, or cholecystectomy rules out this possibility. Furthermore, the intraepithelial presence of parasites confirms tissue invasion by microsporidia. Although a causal relation between microsporidia infection of the biliary tract and AIDS-related cholangitis is difficult to prove, the presence of E. bieneusi in the bile of these patients with AIDS-related cholangitis strongly suggests a causative role, by analogy with intraepithelial biliary cryptosporidiosis and cytomegalovirus infection. Interestingly, biliary cryptosporidium infection was associated with biliary microsporidia infection in two patients who did not excrete cryptosporidium in the feces. Although cryptosporidium and cytomegalovirus-associated infection of the biliary tract is rare, a dual protozoal infection was observed in one third of the patients with AIDS-related cholangitis.
Cholangitis is considered to be a late complication of AIDS3. Two of the eight patients with biliary microsporidia infections belonged to group II of the CDCP classification and had AIDS only months later, suggesting that microsporidia cholangitis should be added to group IV. Indeed, all the patients concerned had very low CD4 cell counts, and most had p24 antigenemia.
It is noteworthy that all eight patients with biliary microsporidiosis were homosexual men, which tends to confirm our view that homosexual behavior is associated with an increased risk of AIDS-related cholangitis4 due to specific sexual practices, as has been proposed for Kaposi's sarcoma17. Indeed, it has recently been established in experiments in animals that bacterial overgrowth of the small intestine can induce histologic inflammation of the portal tract, with bile-duct destruction and proliferation in which cholangiographic abnormalities resemble primary sclerosing cholangitis18; in addition, the process was prevented by antibiotic therapy19,20. Bacterial or parasitic invasion of the biliary system through the portal venous system20 and retrograde infection are two possibilities. Together with the immunodeficiencies related to homosexuality21 and HIV infection, this could explain the frequency of AIDS-related cholangitis in homosexual men.
Unfortunately, we were unable to evaluate the prevalence of cholangitis during the same period that was due to causes other than microsporidia infection. Indeed, the study patients were referred to our unit from other institutions where routine examinations had ruled out cryptosporidiosis and cytomegalovirus infection. However, in a retrospective study of our first 15 cases of AIDS-related cholangitis, biliary infections with cryptosporidia or cytomegalovirus were identified in 9 patients (60 percent)4. Comparable figures have been found in other geographic areas3,22.
Empirical antiprotozoal chemotherapy with metronidazole (1 g twice daily for 15 days) had no clinical benefit with respect to cholestasis or diarrhea, in contrast to its efficacy in microsporidiosis of the small intestine14.
E. bieneusi infection of the biliary tract could account for at least some of the cases of AIDS-related cholangitis that are not explained by cryptosporidium or cytomegalovirus infection. Microsporidia biliary infection usually occurs in patients with severe immunodeficiency, and associated infections with cryptosporidium and microsporidia may be present.
We are indebted to Drs. M. Stern, W. Lowenstein, K. Gillet-Juvin, and J.-L. Dumont for their helpful comments and assistance; to Mr. J.-P. Monnet for his technical help; and to Miss D. Jourdat and Mr. D. Young for their assistance in the preparation of the manuscript.
Source Information
From the Units of Hepatology (S.P., P.B.), Parasitology (C.A.R., J.-F.P.), and Anatomy and Pathology (F.C.), Hopital Necker-Laennec; the Centre d'Endoscopie Digestive Gastrolouvre (P.A.); the Service of Neurohistology, Hopital Pitie-Salpetriere (S.R.); and INSERM U-313 (I.D.-L.) and U-99 (S.P., P.B.) -- all in Paris.
Address reprint requests to Dr. Pol at the Unite d'Hepatologie, Hopital Laennec, 42 Rue de Sevres, 75340 Paris CEDEX 07, France.
References
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