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.

Tropheryma whipplei, a Potential Commensal Detected in Individuals Undergoing Routine Colonoscopy

“…The prevalence of T. whipplei in duodenal biopsy samples was in line with previously described percentages (0 to 4.8%) (13)(14)(15)(16). Others, however, have reported a higher prevalence (7.24%) (17).…”

Reduced Peripheral and MucosalTropheryma whipplei-Specific Th1 Response in Patients with Whipple’s Disease

“…healthy individuals is probably induced by regular contact of the enteric immune system with T. whipplei. Indeed, the presence of T. whipplei-specific IgG Abs in the serum and T. whipplei DNA in the gastrointestinal tract of some healthy individuals (7,9,10) support this hypothesis. Additionally, according to our results, active triathletes, exposed to open watercourses, revealed the highest percentages of T. whipplei-specific CD4 ϩ T cells followed by the age matched, and finally the young subjects.…”

“…whipplei is assumed to be a microorganism present in the environmental soil and water (5). An oral route of acquisition was proposed (6), and in recent studies, T. whipplei DNA was detected in the saliva (7), marginal and subgingival plaque (8), and feces (9) of healthy subjects in which T. whipplei-specific IgG Abs were identified in over 70% of the cases (10). Whipple's disease is very rare despite the almost ubiquitous occurrence of T. whipplei in the environment, suggesting that host factors are necessary to permit an infection.…”

Clinical Manifestations, Treatment, and Diagnosis of Tropheryma whipplei Infections

“…As the bacterium is not evenly distributed over the duodenum, several samples of the duodenum should be obtained in order to avoid sampling bias in patients who do carry the bacterium in their duodenum. In addition, it is also advisable to obtain samples from the gastric antrum, jejunum, and/or ileum (10,51,62,102,111,169).…”

“…Asymptomatic carriers of T. whipplei represent a large reservoir from which other humans might be colonized. The bacterium has been found in various samples, including saliva, urine, blood, cardiac valve, myocardium, synovial fluid, skeletal muscle, stool, skin, lymph node, lung, bronchoalveolar fluid, stomach, spleen, liver, larynx, small bowel, colon, maxillary sinus, cerebrospinal fluid, brain, and aqueous humor samples (18,42,43,45,(49)(50)(51).…”