A case of an AIDS patient infected with the Whipple's disease bacterium, Tropheryma whippelii, is reported. A DNA fragment with sequence specificity for the 16S rRNA gene of the bacterium was detected by PCR in a duodenal biopsy specimen from a 55-year-old male patient with AIDS and diarrhea. The biopsy specimen contained periodic acid-Schiff stain-positive macrophages which did not, however, resemble the sickleform-particle-containing cells characteristic of Whipple's disease. This observation raises two possibilities: either the patient had a coincidence of AIDS and Whipple's disease or Tropheryma whippelii acted as an opportunistic pathogen in this immunodeficient patient. The latter explanation is of interest in light of the ongoing discussion of immunologic abnormalities as predisposing factors for Whipple's disease

Hartmann, M

Maiwald, Matthias

Meier-Willersen, HJ

von Herbay, Axel

Journal of Clinical Microbiology

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Archived at the Flinders Academic Commons http://dspace.flinders.edu.au/dspace/ This is the publisher’s copyrighted version of this article. The original can be found at: http://jcm.asm.org/cgi/reprint/33/5/1354.pdfJOURNAL OF CLINICAL MICROBIOLOGY, May 1995, p. 1354–1356 Vol. 33, No. 50095-1137/95/$04.0010Copyright q 1995, American Society for MicrobiologyDetection of Tropheryma whippelii DNA in a Patient with AIDSMATTHIAS MAIWALD,1* HANS JO¨RG MEIER-WILLERSEN,2† MARTIN HARTMANN,3AND AXEL VON HERBAY4Institute of Hygiene and Microbiology,1 Department of Internal Medicine V,2 Department of Dermatology,3and Institute of Pathology,4 University of Heidelberg, 69120 Heidelberg, GermanyReceived 9 January 1995/Returned for modification 25 January 1995/Accepted 9 February 1995A case of an AIDS patient infected with the Whipple’s disease bacterium, Tropheryma whippelii, is reported.A DNA fragment with sequence specificity for the 16S rRNA gene of the bacterium was detected by PCR in aduodenal biopsy specimen from a 55-year-old male patient with AIDS and diarrhea. The biopsy specimencontained periodic acid-Schiff stain-positive macrophages which did not, however, resemble the sickleform-particle-containing cells characteristic of Whipple’s disease. This observation raises two possibilities: eitherthe patient had a coincidence of AIDS and Whipple’s disease or Tropheryma whippelii acted as an opportunisticpathogen in this immunodeficient patient. The latter explanation is of interest in light of the ongoing discus-sion of immunologic abnormalities as predisposing factors for Whipple’s disease.Whipple’s disease is a chronic multisystem disorder of bac-terial origin (3). It is an uncommon disease, with only 617 casesreported worldwide between 1907 and 1986 (3). As the caus-ative bacterium cannot be cultured, the conventional meansfor diagnosis are histology showing the presence of periodicacid-Schiff stain (PAS)-positive inclusions in histiocytes andelectron microscopy showing rod-shaped bacteria with a typi-cal trilaminar outer cell membrane (3, 15). Characterization ofthe bacterium was successful when PCR with primers for uni-versally conserved regions of the bacterial 16S rRNA gene wasapplied (12, 17). On the basis of its 16S rRNA sequence, thebacterium was described as a new species and the name Troph-eryma whippelii was proposed (12). The natural habitat of thebacterium and its route of infection are currently unknown.However, besides the presence of the causative bacterium,which usually responds promptly to antimicrobial therapy,most patients with Whipple’s disease have an impairment ofimmune function, which is presumed to play a role in patho-genesis (3). As in AIDS, the immune defect in Whipple’sdisease consists of a decreased T-helper/T-suppressor (CD4/CD8) cell ratio during active disease, as well as other immu-nologic abnormalities (9).At present, there is no convincing report of infection causedby the Whipple’s disease bacterium in AIDS patients, althoughtwo previous reports suggested such an association (1, 6). Thereport by Autran et al. (1) was later criticized, because retro-spectively this case was likely the result of infection by Myco-bacterium avium (13), and the report by Jankovic (6), althoughclinically and histologically suggestive, was not confirmed byelectron microscopy. Instead, other opportunistic bacterial in-fections in AIDS, namely those with Mycobacterium aviumcomplex and Rhodococcus equi, were reported to mimic thehistologic appearance of Whipple’s disease by the presence ofPAS-positive inclusions in macrophages (5, 13, 16).We observed a 55-year-old Caucasian man with a history ofhomosexuality and human immunodeficiency virus infectiondiagnosed in 1987. Kaposi’s sarcoma was diagnosed in 1991(Centers for Disease Control and Prevention classificationC2). As tumor progression was slow, he received no antineo-plastic therapy. In 1992, he suffered from recurrent episodes ofdiarrhea, fever, and loss of appetite. Intestinal candidiasis andgiardiasis were diagnosed and responded to therapy with ny-statin and metronidazole. Since January 1993 he again sufferedfrom watery diarrhea, low-grade fever, dysphagia, and weightloss. Stool examinations for Giardia spp., Candida spp., cryp-tosporidia, and nontuberculous mycobacteria and urine exam-inations for cytomegalovirus were negative. In July 1993, en-doscopy was performed. At this time, the hematocrit was 33%,the leukocyte count was 2.96/nl, the CD4 cell count was 65/ml,the CD8 cell count was 310/ml, and the CD4/CD8 ratio was 0.2.Esophagoscopy revealed Candida esophagitis, gastroscopyshowed normal findings, and duodenoscopy showed a whitishappearance of the mucosa. Histology of duodenal biopsiesrevealed some PAS-positive macrophages in the mucosa (Fig.1). Ziehl-Neelsen stain for mycobacteria was negative. How-ever, the cytologic features of the PAS-positive histiocyteswere not diagnostic for sickleform-particle-containing cells,which are the hallmark of Whipple’s disease (15). Thus, thebiopsy was subjected to PCR analysis.A cryopreserved duodenal biopsy specimen obtained by en-doscopy was processed for PCR analysis by standard tech-niques (11). Briefly, the biopsy specimen was digested for 2 hat 568C in 40 ml of lysis buffer (50 mM KCl, 10 mM Tris, 1.5mM MgCl2, 1% Triton X-100, 200 mg of proteinase K per ml)and boiled for 10 min after the addition of 20 ml of a 20%Chelex suspension (biotechnology-grade chelating resin Chelex100, 100-200 mesh, sodium form; Bio-Rad, Richmond, Calif.).Ten microliters of the supernatant was added to the PCRmixture. The Whipple-specific primers pW3FE and pW2RB(12) were used in a modified version (without restriction en-donuclease recognition sites) in a PCR mixture consisting of100 ml containing 50 mM KCl, 50 mM Tris (pH 9.0), 2.0 mMMgCl2, each deoxynucleotide triphosphate at 200 mM, 50 pmolof each primer, and 2.5 U of Taq polymerase (AmpliTaq DNApolymerase; Perkin-Elmer, Norwalk, Conn.). After 70 ml ofmineral oil was added, the PCR was performed in a Perkin-Elmer 480 thermal cycler using initial denaturation at 958C for3 min followed by 40 cycles of denaturation at 958C for 45 s,annealing at 628C for 55 s, extension at 728C for 55 s, and finalextension at 728C for 2 min. The PCR products were analyzed* Corresponding author. Mailing address: Hygiene-Institut der Uni-versita¨t Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg,Germany. Phone: 49-6221-567815. Fax: 49-6221-563943. Electronicmail address: un69mm@genius.embnet.dkfz-heidelberg.de.† Present address: Abteilung fu¨r Chirurgische Endoskopie, Klini-kum der Stadt Mannheim, Theodor-Kutzer-Ufer, 68135 Mannheim,Germany.1354by electrophoresis on 8% polyacrylamide gels as describedpreviously (7) and subjected to direct nucleotide sequencingwith the Perkin-Elmer AmpliTaq Cycle sequencing kit accord-ing to the instructions of the manufacturer.The resulting PCR product had the expected length of theWhipple-specific DNA fragment (Fig. 2). The sequence wasthe same as that reported for the Whipple’s disease bacterium(12), with the exception of one nucleotide position (deletion ofC at position 1160 of the Whipple-specific DNA nucleotidesequence). The same deletion had previously been found inanother patient with confirmed Whipple’s disease (10). Toconfirm this finding, another cryopreserved biopsy specimenand a paraffin-embedded tissue section from the AIDS patient,which were obtained during the same endoscopy, were pro-cessed in separate experiments. Again, PCR products with thesame nucleotide sequence were obtained.The detected DNA fragment from the gene coding for 16SrRNA is considered to be unique to the Whipple’s diseasebacterium (Tropheryma whippelii) (12). Therefore, this is thefirst confirmed report of the Whipple’s disease bacterium de-tected in an AIDS patient. However, interpretation of thisfinding is difficult. The symptoms of this patient, consisting oflow-grade fever, weight loss, and diarrhea, occur both in clas-sical Whipple’s disease (8) and in AIDS enteropathy (5), andin the latter they are associated with a variety of differentpathogens (14). Also, the histologic features of the duodenalbiopsy were not diagnostic, as the number of PAS-positiveinclusions in histiocytes was not nearly as high as that usuallyobserved in cases of Whipple’s disease (15). Therefore, twomain explanations are possible. Either this patient had a merecoincidence of AIDS and infection with the Whipple’s diseasebacterium, which should be a rare event, or Tropheryma whip-pelii has the ability to act as an opportunistic pathogen inimmunodeficiency. The immune defect of AIDS could haveallowed the bacterium to penetrate the epithelial barrier andmultiply in the duodenal mucosa. The latter explanation is ofinterest, as the role of an immune deficit predisposing forFIG. 1. Duodenal biopsy from the patient under study. Some histiocytes with PAS-positive material are present in the lamina propria mucosae (arrows). Theepithelium is intact.FIG. 2. Polyacrylamide gel electrophoresis after PCR amplification from du-odenal biopsies with Whipple-specific primers. Lanes: 1, 100-bp DNA marker; 2,Whipple’s disease (positive control); 3, AIDS patient under study; 4, other AIDSpatient infected with Mycobacterium avium complex; 5, normal duodenum. APCR product of approximately 267 bp is detected in Whipple’s disease (lane 2)and in the AIDS patient under study (lane 3) (arrow).VOL. 33, 1995 NOTES 1355Whipple’s disease has been discussed (4), specific immunologicabnormalities have been observed (3, 9), and Whipple’s dis-ease associated with opportunistic infections has been reported(2, 8, 10).Nucleotide sequence accession number. The GenBank ac-cession number for the nucleotide sequence of the Whipple-specific DNA fragment is M87484 (12).This work was supported by the Deutsche Forschungsgemeinschaft(grant Ma 1663/2-1).REFERENCES1. Autran, B., B. Gorin, M. Leibowitch, L. Laroche, J. P. Escande, J. Hewitt,and C. Marche. 1983. AIDS in an Haitian woman with cardiac Kaposi’ssarcoma and Whipple’s disease. Lancet i:767–768.2. Bassotti, G., M. A. Pelli, R. Ribacchi, M. Miglietti, M. L. Cavalletti, M. E.Rossodivita, P. Giovenali, and A. Morelli. 1991. Giardia lamblia infestationreveals underlying Whipple’s disease in a patient with longstanding consti-pation. Am. J. Gastroenterol. 86:371–374.3. Dobbins, W. O., III. 1987. Whipple’s disease. Charles C Thomas, Publisher,Springfield, Ill.4. Dobbins, W. O., III. 1981. Is there an immune deficit in Whipple’s disease?Dig. Dis. Sci. 26:247–252.5. Gillin, J. S., C. Urmacher, R. West, and M. Shike. 1983. DisseminatedMycobacterium avium-intracellulare infection in acquired immunodeficiencysyndrome mimicking Whipple’s disease. Gastroenterology 85:1187–1191.6. Jancovic, J. 1986. Whipple’s disease of the central nervous system inAIDS. N. Engl. J. Med. 315:1029–1030.7. Maiwald, M., H.-J. Ditton, H.-G. Sonntag, and M. von Knebel Doeberitz.1994. Characterization of contaminating DNA in Taq polymerase whichoccurs during amplification with a primer set for Legionella 5S ribosomalRNA. Mol. Cell. 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Owen, and D. F. Keren. 1983. AIDS with Mycobacteriumavium-intracellulare lesions resembling those of Whipple’s disease. N. Engl.J. Med. 309:1324–1325.14. Smith, P. D., H. C. Lane, V. J. Gill, J. F. Manischewitz, G. V. Quinnan, A. S.Fauci, and H. Masur. 1988. Intestinal infections in patients with the acquiredimmunodeficiency syndrome (AIDS). Etiology and response to therapy.Ann. Intern. Med. 108:328–333.15. von Herbay, A., and H. F. Otto. 1988. Whipple’s disease: a report of 22patients. Klin. Wochenschr. 66:533–539.16. Wang, H. H., D. Tollerud, D. Danar, P. Hanff, K. Gottesdiener, and S. Rosen.1986. Another Whipple-like disease in AIDS. N. Engl. J. Med. 314:1577–1578.17. Wilson, K. H., R. Blitchington, R. Frothingham, and J. A. P. Wilson. 1991.Phylogeny of the Whipple’s disease-associated bacterium. Lancet 338:474–475.1356 NOTES J. CLIN. MICROBIOL.

Detection of Tropheryma whippelii DNA in a patient with AIDS

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Detection of Tropheryma whippelii DNA in a patient with AIDS

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