Abstract

BackgroundThe reservoir of the agent of Whipple disease is unknown. Asymptomatic carriage of Tropheryma whipplei in human stool and saliva is controversial

MethodsStools and saliva specimens from 231 workers at a sewage treatment facility and from 10 patients with Whipple disease, stool specimens from 102 healthy people, and stool specimens from 127 monkeys or apes were tested for T. whipplei DNA by a quantitative real-time polymerase chain reaction with probe detection. Genotyping and culture of T. whipplei–positive samples were performed

ResultsAsymptomatic carriage in stool was found in humans (ranging from a prevalence of 4% in the control group to 12% among a subgroup of sewer workers) but not in monkeys and apes. The T. whipplei load in stool was significantly lower in carriers than in patients with Whipple disease (P<.001). There was a significant prevalence gradient associated with employment responsibilities at the sewage treatment facility: workers who cleaned the underground portion of the sewers were more likely than other workers to carry T. whipplei in stool. Seven of 9 sewer workers tested positive 8 months later. Patients with Whipple disease were significantly more likely to have T. whipplei–positive saliva specimens (P=.005) and had a significantly greater T. whipplei load in saliva (P=.015), compared with asymptomatic stool carriers from the sewage facility. All non–stool carriers had T. whipplei–negative saliva specimens. T. whipplei strains were heterogeneic among sewer workers but identical within individual workers

ConclusionChronic asymptomatic carriage of T. whipplei occurs in humans. Bacterial loads are lower in asymptomatic carriers, and the prevalence of carriage increases with exposure to sewage

Tropheryma whipplei causes Whipple disease, an infectious disorder mainly involving the digestive tract [1, 2]. Since 2000, culture and analysis of the bacterium has allowed a better understanding of the disease it causes [2–4]. Two important but yet unsolved issues are the bacterium’s natural habitat and the route of infection. Analysis of stool samples by polymerase chain reaction (PCR) has detected T. whipplei DNA in patients with Whipple disease [5, 6]. Recovery of T. whipplei from culture of stool from a patient with Whipple disease has demonstrated the presence of viable bacteria, suggesting that the disease could be linked to fecal-oral transmission [7]

T. whipplei DNA has been reported in 6%–11% of stool specimens from people without Whipple disease [8, 9] and in wastewater samples from Germany and Austria, supporting the hypothesis that the bacterium is a soil or water inhabitant [10, 11]. In a recent study involving nested PCR of stool specimens, Schoniger-Hekele et al. [11] found that detection of T. whipplei DNA seemed to be significantly more frequent among sewer workers (16 [25%] of 64) than among control subjects with other diseases (12 [7%] of 178). However, repeat PCR yielded positive results for only 2 of 28 specimens that initially tested positive for T. whipplei

A high prevalence of asymptomatic carriage of T. whipplei in saliva was reported by Dutly et al. [12]. In 2 studies, T. whipplei DNA was detected in saliva samples from 19% and 35% of healthy subjects [12, 13]. However, such high frequencies have not been confirmed by other groups [2, 9, 11, 14, 15]. Recently, it was found that the primers used in previous studies for the detection of T. whipplei also amplified Eikenella corrodens and Actinomyces odontolyticus DNA in saliva, suggesting the possibility of false-positive results [16]. However, amplicon sequencing analysis by Dutly et al. [12] identified 3 internal transcribed spacer (ITS) variants of T. whipplei in asymptomatic carriers, and identical ITS types were observed in different specimens obtained from the same individual. Moreover, because all control specimens were negative for T. whipplei the data from Dutly and colleagues suggest that contamination should be excluded as a possible explanation of the positive PCR results

Little is known about the possibility of T. whipplei carriage or Whipple disease in animals. To our knowledge, only 1 case has been reported, and it involved a zoo-dwelling gorilla that had clinical manifestations similar to those of Whipple disease (i.e., histiocytic colitis) [17]. Granulomatous colitis with histological resemblance to Whipple disease has also been described in dogs [18]

The question of asymptomatic carriage of T. whipplei in humans or other primate species remains unresolved. Here, we report results of T. whipplei–specific PCR of stool and saliva specimens from 231 workers at a sewage treatment facility. We also analyzed stool specimens from 102 people without Whipple disease and from 126 monkeys and apes, using T. whipplei PCR

Subjects, Materials, and Methods

Study subjectsFrom February through September 2006, a total of 231 stool specimens and 231 saliva samples obtained from 231 workers at the municipal sewage treatment facility in Marseille, France, underwent PCR for detection of T. whipplei. The study participants were divided into 3 groups. The first group was composed of 100 workers who used shovels and buckets to manually clean the floors of underground sewers. All of these workers were men, with a mean age (±SD) of 43±9 years (range, 24–61 years). The second group was composed of 111 workers at the same facility who had never worked in the underground part of the sewer; rather, they cleaned the above-ground portions of the sewers or worked at the sewage purification station. A total of 106 were men, and the mean age (±SD) was 40±10 years (range, 20–61 years). The third group was composed of 20 workers from the facility; all had administrative duties, and none had worked in the sewers. Seven persons in this group were men, and the mean age (±SD) was 43±8 years (range, 35–57 years). Workers with a positive PCR result were contacted through their occupational physician (M.T.) and were invited for a specialized consultation (with D.R.)

During the same period, 102 stool samples from 102 control subjects without Whipple disease were tested for T. whipplei by means of PCR. Forty-six control subjects were men, and the mean age (±SD) was 52±23 years. Additionally, 10 patients with a diagnosis of Whipple disease confirmed by our laboratory were also included in the study for quantitative comparisons [19, 20]. For all 10 patients, PCR of stool obtained at the time of diagnosis was positive for T. whipplei. Six patients were men, and the mean age (±SD) was 59±16 years (range, 34–78 years). Nine patients presented with classic Whipple disease, with T. whipplei detected in small-bowel biopsy specimens by means of periodic acid–Schiff staining, immunohistochemical analysis, and/or PCR. The tenth patient presented with uveitis due to T. whipplei PAS staining and PCR of a small-bowel biopsy specimen did not detect T. whipplei However, the pathogen was detected in an aqueous humor specimen by PCR followed by sequencing analysis targeting the 16S RNA sequence, as well as by PCR specific for T. whipplei For 8 patients, a saliva specimen was also available for PCR assay

Fifty stool specimens from 50 macaques, 9 stool specimens from 9 baboons, and 1 stool specimen from 1 gorilla were collected for T. whipplei PCR. In addition, stool specimens gathered in 76 tubes from 62 chimpanzees and 5 gorillas that live in captivity in 12 different groups were also available for T. whipplei PCR. Details regarding the origin of these samples are presented in tables 1 and 2

Table 1

Characteristics of and Tropheryma whipplei polymerase chain reaction findings for nonhuman primates

Table 1

Characteristics of and Tropheryma whipplei polymerase chain reaction findings for nonhuman primates

Table 2

Details of pooled stool specimens collected from 10 groups of chimpanzees and 2 groups of gorillas living in captivity

Table 2

Details of pooled stool specimens collected from 10 groups of chimpanzees and 2 groups of gorillas living in captivity

This study was approved by the local ethics committee. All participants gave informed consent

Stool and saliva PCR forT. whippleiOne gram of stool was obtained for DNA extraction with the QIAamp DNA MiniKit (Qiagen), which was performed in accordance with the manufacturer’s recommendations. Approximately 1 mL of saliva was collected aseptically in a sterile tube from each person. DNA extraction was performed as described above on 200 μL of saliva from each tube. The first T. whipplei PCR (Lightcycler [Roche Biochemicals]), which targeted a 105-bp repeated sequence of the bacterium, incorporated the primer pair TW27F and TW182R and a Taqman probe (27F-182R) into the reaction mix [16]. The PCR mixture included a final volume of 20 μL, with 10 μL of the Probe Master kit (Qiagen), 0.5 μL (10 pmol/μL) of each primer, 5 μL (2 μmol/μL) of probe, 2 μL of distilled water, and 2 μL of extracted DNA. The amplification conditions involved an initial denaturation step at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing and elongation at 60°C for 60 s, with fluorescence acquisition in single mode. After every 5 samples, T. whipplei–negative controls (water, mix, and human samples) were evaluated

If the result of first assay was positive, it was systematically confirmed by a second PCR assay, with a primer pair (TW13F and TW163R) and Taqman probe (13F-163R) targeting a different DNA sequence; the same amplification conditions described above were used. Details about DNA targets, primers, amplicon sizes, and their positions with respect to the full genome of T. whipplei are available in figure 1

Figure 1

DNA targets, primers, probes, and their positions in the complete genome of Tropheryma whipplei for the 2 polymerase chain reactions used in this study

Figure 1

DNA targets, primers, probes, and their positions in the complete genome of Tropheryma whipplei for the 2 polymerase chain reactions used in this study

For quantitative PCR, sequence-specific standard curves were generated using 10-fold serial dilutions of a standard concentration of 106 organisms of the Marseille-Twist T. whipplei strain. The number of transcript copies in each sample was then calculated from the standard curve, using LightCycler software

Stool culture forT. whippleiT. whipplei was cultivated from PCR-positive stool specimens after a decontamination procedure, using a specific axenic medium [7, 21]. Stool specimens were suspended in Rinaldini medium (6.8 g of NaCl, 0.4 g of KCl, 0.15 g of NaH2PO4, 1.0 g of glucose, 2.2 g of NaCOH3, and 0.002 g of phenol red in 1 mL of distilled water) and mixed v/v with glutaraldehyde 2% (Sigma) diluted in sodium bicarbonate (pH 8). After the pellet was vortexed for 5 min and centrifuged at 7500 g for 10 min, it was harvested, washed twice in water, and incubated in 5 mL of specific axenic medium containing ciprofloxacin (1 μg/mL) and amphotericin B (2 μg/mL) in 25-cm2 flasks held at 37°C in a 5% CO2 atmosphere. Each month, 50% of the specific axenic medium was replaced; for the first 3 months, both ciprofloxacin and amphotericin B were added. Thereafter, amphotericin B (2 μg/mL) and trimethoprim (10 μg/mL) were added to the specific axenic medium. Growth was monitored monthly by quantitative real-time PCR, using the protocol described above

Genotyping ofT. whipplei from sewer workersGenotyping of isolates recovered from saliva and stool specimens targeted a 214-bp sequence from an intergenic spacer localized between open reading frames 133 and 134 of the T. whipplei genome, using the primers TWT133F (5′–GCT-G-C-G-C-G-A-A-G-T-A-A-T-TTG) and TWT133R (5′–AGA-T-A-C-A-T-G-C-G-G-A-G-A-T-ACT), and a 219-bp sequence from an intergenic spacer localized between the gene SecA and open reading frame 131 of the T. whipplei genome, using the primers SECA F (5′–TTT-G-T-C-A-T-A-G-G-C-A-T-T-T-C-T-G-TAG) and SECA R (5′–AGA-C-C-T-C-A-C-T-G-T-T-A-T-A-C-G-GAT). PCR was performed in a PTC-200 automated thermal cycler (MJ Research). A total of 5 μL of each DNA solution was amplified in a 25-μL reaction mixture containing 50 pmol/L of each primer; 200 μmol/L each of dATP, dCTP, dGTP, and dTTP (Invitrogen); 1.5 U of HotstarTaq DNA polymerase (Qiagen); 2.5 μL of 10× PCR buffer; and 1 μL of 25 mmol/L MgCl2. The following conditions were used for amplification: initial heat activation at 95°C for 15 min, followed by 40 cycles of denaturation for 30 s at 94°C, annealment for 30 s at 55°C, and extension for 1 min at 72°C. Amplification was completed by holding the reaction mixture for 5 min at 72°C to allow complete extension of the PCR products. PCR products were purified using the MultiScreen PCR filter plate (Millipore) in accordance with the manufacturer’s recommendations. Amplicons were sequenced in both directions, using the BigDye chemistry kit, version 1.1 (Applied Biosystems), on an ABI 3130XL automated sequencer (Applied Biosystems) as recommended by the manufacturer. A previous analysis performed in our laboratory, which involved 49 T. whipplei strains from 39 patients with Whipple disease and 10 asymptomatic carriers, identified 8 genotypes by use of primers TWT133F/133R and 6 genotypes by use of primers SECA F/SECA R (unpublished data)

Statistical analysisStatistical analyses were performed using Epi Info, version 6.04a (Centers for Disease Control and Prevention). A P value of <.05 was considered to be statistically significant

Results

PCR analysis of stool specimensOverall, 23 (6.9%) of 333 persons analyzed had a stool specimen with a positive PCR result. Of the 231 workers at the sewage treatment facility, 19 (8%) had positive PCR results (table 3), compared with 4 (4%) of 102 control subjects (P=.017). Interestingly, positive PCR results were significantly more common among workers with exposure to underground sewers (12 [12%] of 100) than among persons without such exposure (11 [4.7%] of 233; P=.01), including other sewer workers and control subjects (table 4). Of the other sewer workers, 7 (6.3%) of 111 with exposure to above-ground sewers and 0 of 20 with administrative roles had T. whipplei–positive stool specimen. Thus, there was a gradient (from 0% to 6.3% to 12%) with a significant trend toward positivity (P=.04) as workers’ job responsibilities brought them into closer proximity to underground sewers

Table 3

Clinical and epidemiological data for 19 sewer workers with ⩾1 stool specimen in which Tropheryma whipplei was detected by polymerase chain reaction (PCR)

Table 3

Clinical and epidemiological data for 19 sewer workers with ⩾1 stool specimen in which Tropheryma whipplei was detected by polymerase chain reaction (PCR)

Table 4

Tropheryma whipplei carriage and DNA load in the human and nonhuman primate study populations

Table 4

Tropheryma whipplei carriage and DNA load in the human and nonhuman primate study populations

The T. whipplei DNA loads in stool ranged from 85 to 2500 copies/g (mean, 690±860 copies/g) for sewer workers and from 85 to 5000 copies/g (mean, 1800±1800 copies/g) for control subjects and were significantly lower (P<.001 and P=.03, respectively) than the DNA loads for the 10 patients with Whipple disease diagnosed on the basis of our laboratory’s findings (170 to 2.5×106 copies/g; mean [±SD], 9.8×105±1×106 copies/g) (table 4)

Finally, none of the stools from macaques, baboons, chimpanzees, or gorillas tested positive for T. whipplei by PCR (table 4)

PCR analysis of saliva specimensPCR detected T. whipplei in 5 (2.2%) of 231 saliva specimens from sewer workers. Three specimens (3%) were from workers with underground exposure, and 2 (1.8%) were from workers with above-ground exposure only; no worker with an administrative role had a T. whipplei–positive saliva specimen. Furthermore, all PCR-positive saliva samples were from the 19 workers with stool carriage of T. whipplei (table 3). Of the 8 patients with Whipple disease who had a saliva specimen available, 7 (87.5%) had positive PCR results. Thus, PCR-positive saliva specimens were significantly more common among patients with Whipple disease than among asymptomatic stool carriers (P=.005). Additionally, asymptomatic stool carriers were significantly more likely than non–stool carriers to have T. whipplei–positive saliva specimens (5 [26.3%] vs. 0; P<.001)

The mean T. whipplei DNA load (±SD) in saliva samples from sewer workers was significantly lower than that for patients with Whipple disease who had a saliva specimen available (400±300 copies/mL [range, 50–850 copies/mL] vs. 1.7×106±4.7×106 copies/mL [range, 400 to 1.2×107 copies/mL]; P=.015) (table 4)

Consultation and follow-up with sewer workersNineteen workers with a PCR-positive stool specimen underwent follow-up consultation (table 3). Nothing remarkable was found, but 1 worker had chronic arthralgia. Most workers reported having had several diarrhea episodes per year, which is a common finding in this population [22]. Nine had follow-up stool specimens obtained and tested 8 months later, and results for 7 (78%) were positive. Stool specimens from 5 were also tested 1 year after the first screening, all of which were positive for T. whipplei

Recovery of isolates from stool culturesT. whipplei continues to be cultivated from 2 T. whipplei–positive stool specimens 11 months after the date of inoculation

Genotyping of isolates from sewer workersData from amplification and sequencing analysis of the 2 different intergenic spacers were available for 11 sewer workers. A dendogram showing the phylogenetic organization of the genotypes is presented in figure 2. The dendogram demonstrated that the genetic heterogeneity among the tested spacers was wide. For each worker from whom other T. whipplei–positive stool samples were available during the follow-up period, the genotype of T. whipplei from the initial sample was the same as that of isolates from the follow-up samples. Additionally, for each worker from whom T. whipplei isolates were recovered from stool and saliva specimens, the genotype of both isolates was the same

Figure 2

Dendrogram, constructed using the UPGMA method, showing the phylogenetic diversity of Tropheryma whipplei detected in sewer workers (SWs). Sequences from the 2 intergenic spacers were concatenated

Figure 2

Dendrogram, constructed using the UPGMA method, showing the phylogenetic diversity of Tropheryma whipplei detected in sewer workers (SWs). Sequences from the 2 intergenic spacers were concatenated

Discussion

We found asymptomatic carriage of T. whipplei in stool specimens from humans but not in stool specimens from monkeys or apes. We believe our findings are reliable, because all positive PCR results were systematically confirmed by a second PCR assay, and analysis of all negative controls yielded expected results. Moreover, genotyping analysis confirmed all positive samples. An exposure-associated risk factor for carriage exists, as stool carriage of T. whipplei was more prevalent among sewer workers who manually cleaned the underground part of sewers, compared with other workers at the sewage treatment facility and healthy control subjects. There was also a significant prevalence gradient among sewer workers, with a T. whipplei prevalence of 12% among workers with exposure to underground sewers, 6.3% among workers with above-ground exposure only, and 0% among workers with administrative roles. It could be hypothesized that soil- and water-dwelling bacteria, as well as stool-dwelling bacteria, tend to concentrate in sewage, which would explain the high prevalence among sewer workers who cleaned the underground part of the sewer. Furthermore, there was a significantly lower T. whipplei DNA load in stool from asymptomatic carriers, compared with stool from patients with Whipple disease. This could be one of the explanations for the observed difference in the percentage of asymptomatic carriers of T. whipplei between the studies. Indeed, the detection rate may depend on the sensitivity of the PCR assays used. In addition, because specific genes were not used as internal controls to determine the presence of extracted DNA and the absence of PCR inhibitors in analyses of specimens from monkeys and apes, use of an inadequate DNA extraction kit or the presence of PCR inhibitors cannot be excluded as reasons for the negative results observed for these animals. Finally, it is notable that previous PCR-based studies of intestinal biopsy specimens from different domestic animals (20 cattle, 24 pigs, 10 horses, 15 sheep, 13 dogs, 14 cats, and 19 chickens) did not detect T. whipplei DNA [23]

Follow-up analysis showed that asymptomatic carriage of T. whipplei is chronic. It was previously noticed that outdoor professions are predominant among patients with Whipple disease [24]. However, a high prevalence of Whipple disease among sewer workers has never been reported. None of the T. whipplei–positive workers had classic clinical manifestations of Whipple disease, such as arthralgia or chronic diarrhea. Thus, systematic surveillance for T. whipplei in stool specimens is not necessary for sewer workers. General hygiene practices to prevent fecal-oral transmission of pathogens, such as handwashing, are highly recommended for this population. Finally, our data are consistent with the hypothesis that many persons in any given population are exposed to T. whipplei and that some with predisposing immune factors subsequently develop Whipple disease [2, 25]

For several years, the true prevalence of T. whipplei in the saliva of asymptomatic carriers has been unclear. Initial studies reported a high prevalence of carriage [6, 13]. However, other studies have since reported no T. whipplei DNA in saliva specimens from healthy people or a prevalence of <1.5% [2, 9, 11, 14, 16]. These discrepant data may be linked to false-positive results, as primers used in previous studies have been shown to be unspecific [16]. Another hypothesis is that geographical origin may explain the different prevalences: a Swiss team, which reported a high prevalence of T. whipplei among saliva specimens from white people [12], did not detect T. whipplei DNA in saliva specimens from healthy Malaysian people, despite using the same assays for both populations [23]

In our study, it is interesting that all workers with a PCR-positive saliva specimen also harbored T. whipplei in stool. Additionally, the prevalence of salivary carriage of T. whipplei among these stool-positive workers was significantly lower than that for stool-positive patients with Whipple disease. Furthermore, among stool carriers, the DNA load in saliva samples from asymptomatic carriers was significantly lower than that in patients with Whipple disease. As suggested above, this phenomenon may be one of the explanations for the discrepancies observed in the first studies of T. whipplei carriage. However, the lack of primer specificity can alone explain the high prevalence observed in the first reports [16]. Thus, we confirm that asymptomatic salivary carriage of T. whipplei exists, but its prevalence is very low

Genotyping showed a large amount of genetic diversity among T. whipplei isolates recovered from sewer workers. Furthermore, in individual workers, the genotype was the same for organisms recovered from saliva, stool, and other specimens obtained during the follow-up period. Thus, these data confirm that each individual was likely infected by a single clonal strain, although a patient with a possible double infection has been described elsewhere [23, 26, 27]. Our data also suggest that a single clonal T. whipplei strain was not the source of the epidemic in this population; instead, there appeared to be an increased rate of exposure to the source of the bacteria—presumably, human stool. People may be colonized by several strains, with 1 strain becoming predominant, perhaps through the role of the immune system, while others are never or only occasionally detected. Finally, comparative genomic hybridization analysis may be helpful to better characterize T. whipplei strains recovered from asymptomatic carriers. However, to accomplish this, it is necessary to establish a strain from a sewer worker. At the time of writing, T. whipplei continues to be cultivated from cultures of 2 stool samples plated 11 months earlier. On the basis of our experience in culturing T. whipplei these data suggest that the bacterium is still alive and growing in these 2 stool samples [7, 28, 29]

In conclusion, chronic asymptomatic carriage of T. whipplei is found in humans, but the prevalence is low. The bacterial load is lower in healthy carriers, compared with that in patients with Whipple disease. Carriage is more frequent among people with direct contact with sewage. Because Whipple disease is rare, the existence of people with fewer than expected symptoms or with other, as yet unrecognized clinical manifestations of Whipple disease remains unknown

Acknowledgments

We thank the municipal sewage treatment facility in Marseille, France (Société d’Exploitation du Réseau d’Assainissement de Marseille), and the employees who participated in the study; Pascal Pradeau (Institut de médecine aéronautique du service de santé des armées, Brétigny, France) and Diane Agay (Centre de recherche du service de santé des armées, Grenoble, France); and the Crédit Ministériel “Programme Hospitalier de Recherche Clinique” 2006 (Recherche de l’agent de la maladie de Whipple chez le personnel de la Société d’Exploitation du Réseau d’Assainissement de Marseille ainsi que dans le réseau de la communauté urbaine)

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Potential conflict of interest: none reported
Financial support: Programme Hospitalier de Recherche Clinique