Tropheryma whipplei in Children with Gastroenteritis

This bacterium may be an etiologic agent of gastroenteritis.

Tropheryma whipplei, which causes Whipple disease, is found in human feces and may cause gastroenteritis. To show that T. whipplei causes gastroenteritis, PCRs for T. whipplei were conducted with feces from children 2-4 years of age. Western blotting was performed for samples from children with diarrhea who had positive or negative results for T. whipplei. T. whipplei was found in samples from 36 (15%) of 241 children with gastroenteritis and associated with other diarrheal pathogens in 13 (33%) of 36. No positive specimen was detected for controls of the same age (0/47; p = 0.008). Bacterial loads in case-patients were as high as those in patients with Whipple disease and signifi cantly higher than those in adult asymptomatic carriers (p = 0.002). High incidence in patients and evidence of clonal circulation suggests that some cases of gastroenteritis are caused or exacerbated by T. whipplei, which may be co-transmitted with other intestinal pathogens. F or decades, Whipple disease was considered to be a metabolic disorder in humans (1). An accumulation of data, such as antimicrobial drug susceptibilities and observation of atypical bacteria in intestinal macrophages, has suggested that this disease is an infectious disease. Tropheryma whipplei is recognized as the infectious agent responsible for Whipple disease (2). Recent studies using molecular biology (3)(4)(5) and culture-dependent (6)(7)(8)(9)(10) techniques have enabled the T. whipplei genome to be fully sequenced (11,12) and have resulted in development of new culture media (13), selection of highly sensitive primers for quantitative PCR (14), and genotyping (15)(16)(17)(18)(19). The bacterium is found in a viable form in stools of infected patients (9).

Tropheryma whipplei in Children with Gastroenteritis CME ACTIVITY
Medscape, LLC is pleased to provide online continuing medical education (CME) for this journal article, allowing clinicians the opportunity to earn CME credit. This activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of Medscape, LLC and Emerging Infectious Diseases. Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians. Medscape, LLC designates this educational activity for a maximum of 0.5 AMA PRA Category 1 Credits™. Physicians should only claim credit commensurate with the extent of their participation in the activity. All other clinicians completing this activity will be issued a certifi cate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test and/or complete the evaluation at www. medscapecme.com/journal/eid; (4) view/print certifi cate.

Learning Objectives
Upon completion of this activity, participants will be able to: Describe the epidemiology of • Tropheryma whipplei gastroenteritis Identify the bacterial load associated with • T. whipplei gastroenteritis Specify laboratory fi ndings associated with • T. whipplei gastroenteritis Compare clinical fi ndings of • Until recently, T. whipplei was considered a rare bacterium that caused an uncommon disease (2). However, recent studies have confi rmed that T. whipplei is common in stool samples (20,21). T. whipplei DNA has been detected in sewage and is highly prevalent in the feces of sewer workers (12%-26%) (1,20,(22)(23)(24)(25). Moreover, the prevalence of T. whipplei in feces of healthy children 2-10 years of age who lived in rural Senegal (sub-Saharan Africa) was 44% (46/105) (21). These data, together with the genetic heterogeneity of T. whipplei (19), indicate that T. whipplei is a rather common gut bacterium.
We hypothesize that T. whipplei may cause gastroenteritis in children as a result of its primary contact with humans. In a preliminary study, we tested all patients with diarrhea at the University Hospitals in Marseille, France, and determined that this bacterium was found most often in children 2-4 years of age (26).
To investigate whether T. whipplei caused gastroenteritis, we studied the prevalence of T. whipplei DNA in a prospective study of children with diarrhea and controls with diarrhea. We genotyped T. whipplei to identify circulating clones (9) and report overall fi ndings, including data from the preliminary study.

Patients
The study was reviewed and approved by the local ethics committee (agreement no. 07-006). A case-patient was defi ned as a child 2-4 years of age who had 2 positive quantitative PCR results specifi c for 2 T. whipplei DNA sequences, as reported (14,20). From January 2006 through December 2008, we tested 241 stool samples from 241 children with diarrhea at 2 University Hospitals for children in Marseille, France (Timone and Nord Hospitals) using a T. whipplei-specifi c PCR. Samples were obtained from all children in accordance with routine hospital procedures. All stools samples were handled identically. Among 36 children with gastroenteritis and T. whipplei DNA in stools, 11 stool specimens from 10 children were obtained after their recovery from diarrheal illness.
Five stool specimens were tested ≈15 days later, and 6 were sampled ≈1 month later. Eight serum samples from children with gastroenteritis and positive PCR results for T. whipplei were tested retrospectively for T. whipplei by Western blot analysis. Data for patients with a defi nite diagnosis of Whipple disease and positive PCR results for T. whipplei PCR in stools at the time of diagnosis and data for adult asymptomatic carriers of T. whipplei in stools, which have been reported (14,20), were also included for quantitative comparisons.

Controls
All T. whipplei-infected case-patients were compared with 67 gastroenteritis case-patients of the same age. These 67 children had negative results for T. whipplei in stool specimens; epidemiologic, clinical, and biologic features were available for these children.
Forty-seven stool specimens from children 2-4 years of age without gastroenteritis were also tested for T. whipplei by using PCR. Twenty-fi ve children (11 girls and 14 boys) were from kindergarten classrooms at the university hospitals. Samples were also obtained from 10 children (4 boys and 6 girls) hospitalized at Timone Hospital for surgery and from 12 children (5 boys and 7 girls) who visited the Emergency Department of Nord Hospital.
Twenty-fi ve serum samples obtained from children 2-4 year of age with gastroenteritis and T. whipplei-negative results in stool specimens were tested retrospectively. Twenty control serum samples were obtained from 20 children 1-36 months of age with a disease other than gastroenteritis. All serum samples were from children hospitalized in the 2 University Hospitals. These samples were obtained for routine management of these patients, not specifi cally for our study.

Diagnostic Procedures
Bacteria, viruses, and Giardia duodenalis were detected by using standard methods. Stool specimens were plated onto Hektoen, Campylosel, and Yersinia cefsulodin-irgasan-novobiocin agar plates (bioMérieux, Marcy L'Etoile, France). Plates selective for Campylobacter spp. were incubated under microaerophilic conditions; all other media and samples were incubated in ambient air. Temperature of incubation was 37°C, with the exception of Yersinia agar, which was incubated at 30°C. Length of incubation was 5 days. For virus tests, stool specimens were tested by using a chromatographic immunoassay with a VIKIA Rota-Adeno Kit (bioMérieux) kit and electron microscopy with negative staining, which enabled detection of rotavirus, adenovirus, calicivirus, astrovirus, Norwalk virus, coronavirus, and enterovirus. G. duodenalis was detected by PCR, as described (27).
If the result of the fi rst PCR was positive, it was systematically confi rmed by a second PCR with a second set of primer pairs: TW13 forward (5′-TGA GTGATGGTAGTCTGAGAGATATGT-3′) and TW163 reverse (5′-TCCATAACAAAGACAACAACCAATC-3′). This second PCR used a Taqman probe (13 forward-163 reverse, 5′-6-FAM-AGAAGAAGATGTTACGGGTTG-TAMRA-3′) specifi c for a different 150-bp sequence, as described elsewhere; the same amplifi cation conditions described above were used. For quantitative PCR, sequence-specifi c standard curves were generated by using 10-fold serial dilutions of a standard concentration of 10 6 microorganisms of the Marseille-Twist T. whipplei strain. The number of transcript copies in each sample was then calculated from the standard curve by using LightCycler software.
Genotyping of T. whipplei from stool specimens was performed as reported (19). This analysis was specifi c for 4 highly variable genomic sequences (HVGS) and used primers TWT133 forward and reverse for HVGS 1, primers ProS forward and reverse for HVGS 2, primers SECA forward and reverse for HVGS 3, and primers TWT183 forward and reverse for HVGS 4. PCR was performed in a PTC-200 automated thermal cycler (MJ Research, Waltham, MA, USA), as reported (19).
Serologic assays were performed by using Western blotting as described (29). Before blotting, protein concentration was determined by using a commercial reagent (Bio-Rad, Hercules, CA, USA). T. whipplei Twist proteins were resuspended in Laemmli buffer (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France) containing 100 mmol/L dithiothreitol to obtain a fi nal protein concentration of 0.5 μg/μL. The protein lysate was heated for 5 min at 100°C. Five micrograms of native protein was loaded into wells of a 7.5% polyacrylamide gel, and proteins were resolved by sodium dodecyl sufate-polyacrylamide gel electrophoresis.
Proteins were then transferred to nitrocellulose membranes (Transblot Transfer Medium, Pure Nitrocellulose Membrane, 0.45 mm; Bio-Rad) over a 2-hour period by using a semidry transfer unit (Hoeffer TE 77; GE Healthcare, Little Chalfont, UK). Membranes were immersed in phos-phate-buffered saline supplemented with 0.2% Tween 20 and 5% non-fat dry milk (blocking buffer) for 1 h at room temperature and incubated with primary serum (dilution 1:1,000 in blocking buffer) for 1 h at room temperature. Membranes were then washed in triplicate with phosphatebuffered saline-Tween 20, and immunoreactive spots were detected by incubating the membranes for 1 h at room temperature with peroxidase-conjugated goat anti-human antibody (Southern Biotech, Birmingham, AL, USA) diluted 1:1,000 in blocking buffer. The fi rst screening was performed by testing for all immunoglobulins (Igs). Positive cases were then tested to separately detect IgG and IgM. Detection was performed by using chemiluminescence (Enhanced Chemiluminescence Western Blotting Analysis System; Amersham Biosciences, Uppsala, Sweden) with an automated fi lm processor (Hyperprocessor; GE Healthcare).

Statistical Analysis
Data were analyzed by using EpiInfo software version 3.4.1 (Centers for Disease Control and Prevention, Atlanta, GA, USA). Proportions were compared by using the Yates χ 2 corrected test or Fisher exact test. Continuous variables were compared by using analysis of variance or the Mann-Whitney/Wilcoxon 2-sample test when data were not normally distributed. Signifi cance was defi ned as p<0.05.

Results
A total of 241 children 2-4 years of age with gastroenteritis were tested, and samples from 36 (15%) were positive for T. whipplei. In 2006, 2007, and 2008, the infection rates for T. whipplei were comparable: 12/78 (15.4%), 10/72 (14%), and 14/91 (15.4%), respectively. No seasonal variation was observed. None of the children in the same age control group without diarrhea had samples positive for T. whipplei (0/47; p = 0.008). High bacterial loads (>10 4 /g of stool) were observed in 64% (23/36) of the T. whippleipositive children. Such high loads have not been observed in chronic carriers, but they were comparable to the levels for patients with Whipple disease (14). Bacterial load in stools ranged from 170 to 1.5 × 10 6 /g (mean ± SD 1.5 × 10 5 ± 3.6 × 10 5 ) for children with gastroenteritis versus 85 to 2.5 × 10 6 /g (mean ± SD 5.5 × 10 5 ± 8.3 × 10 5 ) for patients with Whipple disease (p = 0.1). Only 1 postdiarrheal stool specimen was slightly positive 15 days later and had a bacterial load <85/g of stool; the bacterial load was 2 × 10 4 at the time of diarrhea. Stool samples obtained from the same patient 1 month later were negative by PCR.
Genotyping of T. whipplei was performed for 34 children with diarrhea. A dendogram showing phylogenetic organization of genotypes is shown in Figure 1. We observed genetic heterogeneity in sequences associated with gastroenteritis and identifi ed 12 new genotypes. One useful fi nd-ing was that genotype 3 was detected in 10 of the 34 children. Using Western blotting, we found that case-patients with Whipple disease were signifi cantly more likely to be seropositive than controls with diarrhea ( Figure 2) for IgG (7/8 vs. 5/25; p = 0.001) and IgM (7/8 vs. 1/25; p<0.001) and than controls without gastroenteritis for IgG (5/20; p = 0.004) and IgM (1/20; p<0.001).
Clinical and biological features of the 36 PCR-positive children 2-4 years of age with diarrhea were compared by retrospective review with those of matched 67 control children of the same age who had gastroenteritis but were negative for T. whipplei by PCR. Results are summarized in Table 1. Among children with positive PCR results, the proportion of girls and boys was equal. T. whipplei PCR-positive patients with diarrhea had a milder illness than PCR-negative patients. Length of rehydration required, duration of fever, and duration of hospitaliza-tion were signifi cantly shorter for the patients infected with T. whipplei (p = 0.003, p = 0.003, and p = 0.01, respectively). Moreover, the level of C-reactive protein was signifi cantly lower incase-patients (p = 0.03). Patients with T. whipplei were less likely to experience anorexia (p = 0.03); however, their weight loss was signifi cant (p = 0.045). Another signifi cant difference was increased contact with sand boxes for case-patients (p = 0.002); however, the size of the group analyzed was small, and these data should be confi rmed.

Discussion
T. whipplei has been identifi ed by PCR in stools of persons without Whipple disease (20). The source of T. whipplei is unknown, but data suggest that it the infection may result from fecal-oral or oral-oral transmission (2,20). T. whipplei is excreted in a live form by patients with Whipple disease (9), and T. whipplei DNA is found in stool samples of healthy persons (22). This bacterium is commonly found (1) 14 13 13 11 (1)  in sewers (20,23) and has also been detected in sewage (25). Therefore, it appears that T. whipplei is much more common than previously believed. A total of 3.8% (7/299) of adult controls in our study were PCR positive for T. whipplei (14). The bacterial load of T. whipplei in stools is much lower in asymptomatic carriers than in patients with Whipple disease (14,20). We believe that our data provide strong evidence to support our initial hypothesis that T. whipplei causes mild gastroenteritis in children 2-4 years of age. All of our case-patients had 2 quantitative PCR-positive results, and genotyping of T. whipplei from 32 children enabled us to identify 12 new genotypes. Our data exclude the possibility of PCR contamination. We also found bacterial loads higher than those in previous reports of chronic carriers (20). However, these loads were comparable with those for patients with active Whipple disease (14). These high loads suggest that gastroenteritis in the children in our study was associated with active T. whipplei replication.
The absence of T. whipplei DNA in stools after patient recovery from diarrheal illness strongly suggests that detection of T. whipplei DNA is associated with acute gastroenteritis rather than carriage, which is usually chronic (20).
Moreover, 1 genotype (genotype 3) predominated, causing one third of the total number of cases.
Serologic analysis by Western blotting for T. whipplei (29) showed that all children who were PCR positive for T. whipplei had IgM against T. whipplei, which suggested that recent seroconversion had occurred. The prevalence of antibodies in case-patients was higher than that in controls. Comparison of the prevalence of antibodies for control children (22%) with preliminary prevalence data for persons 61 ± 3.6 years of age (45%) (30) showed that the difference is signifi cant (p<0.001); thus, possible acquired immunity to infection with T. whipplei may explain why this bacterium causes diarrhea in children, but not in most adults.
We suspect that T. whipplei infection is contagious and transmitted by the fecal-oral route in children 2-4 years of age with other enteric pathogens. We previously reported an association between G. duodenalis infection and Whipple disease (27). As with other organisms such as Helicobacter pylori, T. whipplei may also be transmitted through saliva because it has been found in saliva of asymptomatic carriers and patients with Whipple disease (20). Therefore, we hypothesized that primary infection with T. whipplei in children may result in gastroenteritis, especially when associated with other intestinal pathogens. We have provided several lines of evidence that T. whipplei is causing or exacerbating gastroenteritis. The incidence of T. whipplei DNA in stools of children 2-4 years of age with gastroenteritis was higher than that in the control group; these infected children also have higher levels of antibodies. That healthy children of the same ages were not infected with T. whipplei also suggests that primary infection is symptomatic. Sociodemographic differences between the 2 groups could theoretically explain our fi ndings. However, most of our patients and controls were from the same geographic area, which enabled us to rule out this hypothesis. We identifi ed 1 clone (genotype 3) in 10 children, which indicates that this clone is circulating in our population. The association we found between T. whipplei and other pathogens transmitted by the fecal-oral route supports the conclusion that T. whipplei and other intestinal pathogens have a common source of infection and that they are often co-transmitted (31,32). However, for unknown reasons, T. whipplei could replicate in children with lowgrade chronic infections without causing diarrhea. That T. whipplei-infected patients were more likely to have co-infections than patients infected with other pathogens may also indicate that T. whipplei may decrease below molecular detection limits when diarrhea resolves. However, higher levels of IgM against T. whipplei in case-patients suggest infection with this bacterium. Moreover, an in vivo animal model of oral infection by T. whipplei has shown a pathogenic effect only in mice with previously infl amed colonic tissues (D. Raoult et al., unpub. data). Thus, infl amed colonic tissues may also explain the frequency of co-infections with common pathogens observed in persons with T. whipplei-positive gastroenteritis.
We provide evidence that T. whipplei is commonly associated with gastroenteritis in children. We also suggest that other studies should be performed to evaluate the role of this bacterium and its prevalence in patients with gastroenteritis because it is present worldwide. All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required.