Detection of Tropheryma whipplei DNA in feces by PCR using a target capture method

Abstract

Whipple's disease is a rare multisystemic bacterial infection with variable clinical manifestations. For decades, the laboratory diagnosis was based on the demonstration of periodic acid Schiff-positive inclusions in macrophages of gastrointestinal biopsies. PCR has improved the diagnosis of Whipple's disease due to its increased sensitivity compared to histopathological analysis. To avoid invasive procedures for taking specimens, we have investigated the possibility of detecting Tropheryma whipplei DNA in feces rather than in biopsies or gastric aspirate of patients with and without Whipple's disease. Total bacterial DNA was isolated from stool specimens using Qiagen columns followed by a T. whipplei-specific hybridization step with a biotinylated capture probe and streptavidin-coated magnetic particles. The captured DNA was then amplified using the same seminested PCR targeting the 16S rRNA gene of the organism that had been applied to other specimens without capturing. For five of eight patients with Whipple's disease, duodenal biopsies and stool samples were PCR positive, whereas for the three other patients, both specimens were PCR negative. Of 84 patients without Whipple's disease, 75 tested negative in the duodenal biopsy and in the stool sample. For four, both specimens were positive. Five patients tested positive in the stool sample but not in the biopsy. However, for three of these five patients, the gastric aspirate had been PCR positive, indicating that the stool PCR result was true rather than false positive. Compared to PCR from duodenal biopsies, stool PCR has a sensitivity of 100% and a specificity of 97.3%. Additionally, 15 PCR-positive and 22 PCR-negative stool samples were extracted using the Invisorb Spin Stool DNA kit. The simplified stool extraction showed 93.3% sensitivity and 95.5% specificity compared to the target capture method. We conclude that PCR with stool specimens with either extraction method is a sensitive and specific diagnostic tool for the detection of T. whipplei DNA and one not requiring invasive sampling procedures.

FIG. 1.

Schematic overview of the positions of primers and probes used for amplification and capturing, respectively. Positions are shown according to the rRNA genes of E. coli.