Potential Role for Urine Polymerase Chain Reaction in the Diagnosis of Whipple's Disease

Abstract

Background: Whipple's disease (WD) is a rare infection with Tropheryma whipplei that is fatal if untreated. Diagnosis is challenging and currently based on invasive sampling. In a case of WD diagnosed from a kidney biopsy, we observed morphologically-intact bacteria within the glomerular capsular space and tubular lumens. This raised the questions of whether renal filtration of bacteria is common in WD and whether polymerase chain reaction (PCR) testing of urine might serve as a diagnostic test for WD.

Methods: We prospectively investigated urine samples of 12 newly-diagnosed and 31 treated WD patients by PCR. As controls, we investigated samples from 110 healthy volunteers and patients with excluded WD or acute gastroenteritis.

Results: Out of 12 urine samples from independent, therapy-naive WD patients, 9 were positive for T. whipplei PCR. In 3 patients, fluorescence in situ hybridization visualized T. whipplei in urine. All control samples were negative, including those of 11 healthy carriers with T. whipplei-positive stool samples. In our study, the detection of T. whipplei in the urine of untreated patients correlated in all cases with WD.

Conclusions: T. whipplei is detectable by PCR in the urine of the majority of therapy-naive WD patients. With a low prevalence but far-reaching consequences upon diagnosis, invasive sampling for WD is mandatory and must be based on a strong suspicion. Urine testing could prevent patients from being undiagnosed for years. Urine may serve as a novel, easy-to-obtain specimen for guiding the initial diagnosis of WD, in particular in patients with extra-intestinal WD.

Keywords: Tropheryma whipplei; Whipple’s disease; electron microscopy; fluorescence in situ hybridization; real-time PCR.

Figure 1.

Histopathologic findings in the kidney biopsy of the index patient. (A) Periodic acid–Schiff (PAS): few macrophages in the interstitium with PAS-positive particles (Tropheryma whipplei) within the cytoplasm (arrow). (B) Periodic acid–Schiff: glomerulus with numerous, strongly PAS-positive, sickle-shaped particles (Tropheryma whipplei) within the urinary space and close to several visceral and parietal epithelial cells, as well as in the cytoplasm of some distal tubular epithelial cells (bottom right).

Figure 2.

Electron microscopic findings in the kidney biopsy of the index patient. (A and B) Numerous electron-dense rod-shaped structures (Tropheryma whipplei) in the urinary space outside the capillary lumens next to the podocytes, bounded by a trilaminar cell wall. (C) Several T. whipplei bacteria in the urinary space and occasionally (arrows) within electron-dense subepithelial humps, surrounded by the cytoplasm of a podocyte. (D) Few T. whipplei bacteria in the lumen of a proximal tubule. Abbreviations: BB, brush border; BM, basement membrane; CL, capillary lumens; E, erythrocytes; H, electron-dense subepithelial humps; M, mesangium; P, podocytes; TC, tubular cell; TL, lumen; TW, Tropheryma whipplei; US, urinary space

Figure 3.

Tropheryma whipplei detected in the kidney biopsy of the index patient by FISH and immunohistochemistry. (A) Overview of the tissue sample showing the tissue background in green, T. whipplei (detected by REWHIP_Cy3 [26]) in orange, and nucleic acid stain DAPI in blue. (B) Inset B from panel A at a higher magnification, showing a cluster of T. whipplei cells in the tissue. (C) Immunohistochemistry of the identical microscopic view on the identical slide. Immunohistochemistry was performed with specific anti–T. whipplei antibodies. Antibody-stained regions shown in brown. (D) Overlay of the FISH signals from panel B in red and the DAPI-stained host cell nuclei in blue, with the immunohistochemistry image in panel C shown here in in black and white and the immunostain-positive region in gray. Note that only part of the antibody-stained region shows FISH signals. A FISH signal is only present in bacterial cells that contain ribosomes and are, therefore, presumably active at the time of sampling. A combination of FISH and immunohistochemistry, as shown here, may therefore provide therapy-relevant information that cannot be otherwise obtained. Abbreviations: DAPI, nucleic acid stain 4′,6- diamidino-2-phenylindole dihydrochloride; FISH, fluorescence in situ hybridization; REWHIP_Cy3, Tropheryma whipplei–specific FISH probe.

Figure 4.

FISH of the kidney biopsy of the index patient and of a urine sample from a different patient showing morphologically-intact Tropheryma whipplei bacteria. (A) Overview of the kidney tissue of the index patient with the tissue background in green, T. whipplei (detected by REWHIP_Cy3 [26]) in orange, and the nucleic acid stain DAPI in blue. (B) At higher magnification, several T. whipplei bacteria are visible in tubules. (C) Inset from panel B at a higher magnification, showing single, morphologically-intact T. whipplei bacteria. (D) Urine sediment from another patient, showing morphologically-intact T. whipplei bacteria stained with REWHIP_Cy3 [26] (orange), among other bacteria with the pan-bacterial probe EUB338_FITC [25] (green), and the nucleic acid stain DAPI (blue). (E) Identical microscopic view as D, showing only the nucleic acid stain DAPI in black and white. (F) Identical microscopic view as D, showing only pan-bacterial probe EUB338_FITC [25] in black and white. (G) Identical microscopic view as D, showing only REWHIP_Cy3 [26] in black and white. Abbreviations: DAPI, 4′,6- diamidino-2-phenylindole dihydrochloride; FISH, fluorescence in situ hybridization; REWHIP_Cy3, Tropheryma whipplei–specific FISH probe.