Tropheryma whipplei in Patients with Pneumonia

This bacterium may be an etiologic agent of pneumonia.

T ropheryma whipplei is a bacterium widely known to be associated with Whipple disease (WD), which is characterized by various clinical signs such as diarrhea, weight loss, lymphadenopathy, and polyarthritis (1). Furthermore, clinical features such as blood culture-negative endocarditis and neurologic lesions have been reported (2). Until 1991, WD diagnosis was based essentially on histopathologic observations, characterized by positive periodic acid-Schiff stained inclusions within intestinal macrophages (3) and electron microscopy, which has shown the microbiologic etiology of the illness (4).
The fi rst molecular identifi cation was made by Wilson et al. in 1991 (the bacterium was uncultured at that time) by using broad-range primers targeting the 16S subunit of rDNA extracted from infected duodenal tissue of a patient with WD (5). The bacteria were thus classifi ed within the family Actinomycetes (classifi cation was based on 16S rDNA sequence analysis), and the name Tropheryma whippelii was proposed (5,6); the bacterium was later modifi ed to Tropheryma whipplei by La Scola et al. (7). Investigators then developed several molecular PCRbased methods for the diagnosis of WD. Most of these methods use probes or primer sets targeting parts of the 16S or 23S rDNA genes (8).
Successful cultivation of the bacterium by Raoult et al. (9) has enabled the adoption of novel diagnostic tools, such as immunohistochemistry and serologic tests (10)(11)(12). Three years after successful cultivation, the genomes of 2 T. whipplei strains (T-W08/17, GenBank accession no. NC004551, and Twist, GenBank accession no. NC004572) have been fully sequenced (13,14). Completion of the sequencing of these 2 genomes has enabled the design of highly specifi c and sensitive primer pairs that target repeated DNA sequences unique to the T. whipplei genome (15).
These major advances have enabled the identifi cation of T. whipplei DNA in various specimens such as saliva and stools from patients with WD, as well as from asymptomatic carriers, which suggests that the localization of the bacterium is not restricted to the digestive tract and that other organs might be affected (2,(16)(17)(18). More recently, detection of DNA from T. whipplei in bronchoalveolar lavage (BAL) samples from a patient with pneumonia suggests that the bacterium might be involved in respiratory diseases (19). We show that T. whipplei could be a potential infectious agent for patients admitted to intensive care units (ICUs) and that it can be found in the BAL samples of patients in ICUs.

Materials and Methods
All case-patients (hereafter patients) were admitted to 1 of 3 ICUs in Marseille, France (1 medical ICU and 2  (20) in the 3 ICUs. The samples were then transported at room temperature to the microbiology laboratory of Timone Hospital, Marseille, France, and preserved at -20°C until handling. Ventilator-associated pneumonia, community-acquired pneumonia, and aspiration pneumonia were defi ned as previously described (21)(22)(23). DNA was extracted in a MagnaPure LC workstation (Roche Diagnostics, Meylan, France) by using the MagNa Pure LC DNA Isolation Kit II (Roche Diagnostics). Pellets from BAL fl uid samples were mixed with 200 μL of lysis buffer and 50 μL of proteinase K, incubated overnight at 56°C, disrupted for 1 min with glass beads in a MagNa Lyser (Roche Diagnostics), and then processed on the Magnapure LC workstation by the manufacturer's recommendations. When a BAL sample was cell-poor, to concentrate the bacterial cells, we centrifuged 500 μL of BAL sample for 15 min at maximum speed, discarded 400 μL of the supernatant, then resuspended the pellet in the remaining 100 μL; the extraction procedure was continued as described above.
Standard PCR was performed by using the eubacterial broad-range 16S rDNA primer set 536F: 5′-CAGCAGCCGCGGTAATAC-3′ and rp2: 5′-ACGGC TACCTTGTTACGACTT-3′ (Eurogentec, Seraing, Belgium). PCR was performed in an ABI Themocycler (Applied Biosystems, Courtaboeuf, France).The amplifi cation was done in a 50-μL fi nal volume containing 5 μL extracted DNA, 1X PCR buffer (5 μL), 2 μL of 25 mM MgCl 2 , 200 μM of each dNTP, 0.2 mM of each 536F and rp2 primer, and 1 unit of HotStar Taq DNA Polymerase (QIAGEN, Courtaboeuf, France). Amplifi cation started with an initial incubation at 95°C for 15 min to denature DNA and activate polymerase enzymes, followed by 35 cycles of heating at 95°C for 1 min, annealing at 62°C for 30 s, and extension at 72°C for 90 s. Amplifi cation ended with an extension step at 72°C for 10 min. The PCR products were purifi ed by using the Nucleo-Fast 96 PCR Kit (MACHEREY-NAGEL, Hoerdt, France) as defi ned by the supplier, and 4 μL of purifi ed PCR product was sequenced in 20 μL fi nal volume containing sequencing buffer, 3.2 pmole of forward (536F) or reverse (rp2) primer, 3 μL of Big Dye Terminator V1.1 mix (Applied Biosystems), and 8 μL of deionized water. Sequencing reactions were purifi ed by using Sephadex Gel-Filtration (Sigma-Aldrich Chimie, Saint Quentin Fallavier, France), and the purifi ed products were sequenced on an ABI PRISM 3130xl genetic analyzer (Applied Biosystems). Sequences obtained were analyzed with Autoassembler software and compared with those available in the GenBank database by using the BLAST program (www.ncbi.nlm.nih.gov/BLAST).
When the BAL fl uid was polymicrobial, purifi ed PCR products were cloned into PCR II TA cloning vector (Invitrogen, Cergy Pontoise, France) by using 3 μL of purifi ed PCR products from the previous step as recommended by the manufacturer, and 56-64 white colonies were screened for each specimen. The cloned inserts were amplifi ed with M13 primers set (M13F: 5′-GTAAAACGACGGCCAG-3′, M13R: 5′-CAGGAAACAGCTATGAC-3′) and sequenced as described above.
Genotyping of T. whipplei detected in the BAL fl uid samples was conducted as described previously (24). Each of the 4 highly variable genomic sequences (HVGSs) obtained from each specimen was compared with those available in the GenBank database and our internal laboratory database to determine their corresponding genotype. The combination of the 4 HVGSs was then analyzed to defi ne the genotype of the bacteria.
Serologic assays were performed by Western blotting. The T. whipplei Twist strain was cultivated in axenic medium as previously reported (25). Native and deglycosylated samples obtained from the total bacterial extract were prepared for sodium dodecyl sulfate-polycrylamide gel electrophoresis. The assay was performed to test all immunoglobulins (Ig), total (IgT), including IgG, IgM, and IgA heavy and light chains, as well as to test IgG, IgM, and IgA separately, as previously described (26). Detection was performed by using chemiluminescence (ECL Western Blotting Analysis System) and an automated fi lm processor (Hyperprocessor, GE Healthcare, Buckinghamshire, UK). To quantify Western blot bands, we scanned fi lms with an Image Scanner III (GE Healthcare). We performed image analysis by using GelEval 1.21b FrogDance software and ImageJ software (http://rsb.info.nih.gov/ij).

Results
Bacterial DNA of T. whipplei was detected in 6 of 210 BAL fl uid samples by standard or quantitative PCR ( Table 1). The patients' ages ranged from 39 to 73 years (mean ± SD 56.83 ± 14.01 years), and all were men ( Table  2). One of those 6 specimens (no. 5) was positive for the bacterium in both standard and quantitative PCR assays. For this patient, PCR with broad-range primers showed that T. whipplei was the only bacterium identifi ed. Moreover, both quantitative PCR assays showed a high level of the bacterium in this specimen (cycle threshold = 20 with Whi3 probe and cycle threshold = 21 with Whi2 probe; 5.10 5 copy [ Table 1]). This patient was immunocompromised and had community-acquired pneumonia and septic shock ( Table 2). He was admitted to the medical ICU for septic shock and acute respiratory distress syndrome complicating community-acquired pneumonia (Figure). He received chemotherapy for diffi cult-to-treat mediastinal lymphoma. A lobectomy on his upper right lung had been done 1 year before admission for the lymphoma. Lung infi ltrates were present when he was admitted to the hospital. The patient was febrile (39.2°C) and hypoxemic (partial pressure of oxygen in arterial blood [PaO 2 ] 120 mm Hg for a fraction of inspired oxygen [FiO 2 ] at 1), and pancytopenia was evident after initial examination. He received blood products during his ICU stay (9 packed erythrocytes, 4 fresh frozen plasma, 3 platelet transfusions). He was empirically treated by using ticarcillin/clavulanic acid and erythromycin. Hemodynamic status improved rapidly, and administration of vasopressors was stopped by day 2. The patient was fi nally extubated at day 7 and discharged from the ICU on day 10. He fully recovered after completing a treatment regimen of imipenem, amikacin, and vancomycin.
Specimens nos. 10, 82, 86, 183, and 209 from 5 other patients were positive for T. whipplei in quantitative realtime PCR by using the Twhi3 probe and primer pairs and successfully confi rmed by using the Twhi2 probe and primer pairs ( Table 1). The bacterial load in each specimen is shown in Table 1. Standard cultures were negative; 10 4 CFUs were used as the cutoff. The 16S rDNA assay yielded 50 bacteria representing 46 species (Table 1). Of these 46 bacterial species, 15 had <97% 16S rRNA sequence identity with the sequences available in the GenBank database, which suggests a potentially new bacterial species (Table 1). However, despite the high number of the clones screened for these patients, this approach was unable to detect DNA of T. whipplei. This result could be explained by the polymicrobial aspect of these specimens caused by the presence of oral bacterial fl ora, which was proven by the high number of bacteria identifi ed from each specimen ( Table 1). Patients 10, 82, 86, and 183 had a typical buccal fl ora. Two of these 4 patients had aspiration pneumonia; the remaining 2 had ventilator-associated pneumonia.
The patient from whom specimen no. 209 was collected had fl ora of an apparent environment and water origin, but aspiration pneumonia likely developed.
Genotyping of T. whipplei was successfully performed for 4 specimens (specimen nos. 5, 10, 82, and 86). Three new T. whipplei genotypes were identifi ed in specimen nos. 5, 82, and 86. T. whipplei genotype 3 was isolated from specimen 10; this genotype is currently identifi ed in patients with endocarditis caused by T. whipplei and in patients with digestive WD (24). Another new genotype of T. whipplei was identifi ed in specimen no. 183. In this sample, because of the low level of the bacterium, only 2 HVGSs (HVGS1 and HVGS3) were successfully amplifi ed. However, after analysis of the HVGS1 sequence, we concluded that it was a new genotype different from those previously identifi ed. Serologic assays showed no immunoreactivity in the serum of patients whose BAL samples were positive for T. whipplei in molecular assays.

Discussion
We report the use of broad-range primer-based PCR amplifying the 16S rDNA gene followed by cloning and sequencing to identify bacteria associated with pneumonia occurring in mechanically ventilated ICU patients. These results agree with previously published studies (19,27,28) of cystic fi brosis patients, which showed that unusual microbiologic agents can be responsible for pneumonia. The present study shows that these unusual agents of pneumonia can also be identifi ed in mechanically ventilated ICU patients.
Our results show that T. whipplei is an etiologic pathogen in pneumonia. We tested samples from all patients positive for T. whipplei twice by RTq PCR using different primer pairs and probes. In the broad-range PCR-based assay, samples from the fi rst patient were positive for T. whipplei by direct amplifi cation and sequencing of the 16S rDNA gene. Together with the published work of Harris et al., this positive result prompted us to test systematically for T. whipplei in all BAL samples collected (19). Indeed, as we were conducting our study, Harris et al. reported the detection of T. whipplei in the sputum of a child with pneumonia (19). Moreover, we were able to genotype T. whipplei in 5 specimens (i.e., 4 further amplifi cations for nos. 5, 10, 82, and 86 and 2 other amplifi cations for no. 183). Serologic assays showed no immunoreactivity in the serum of the patients of interest. In this age group (61 ± 3.6 years), the percentage of positivity by Western blot is 45% (F. Fenollar, D. Raoult, unpub. data). The absence of immunoreactivity in serum of patients whose BAL fl uid was positive for T. whipplei in molecular assays shows that T. whipplei was probably not present in those patients before the current pneumonia developed. Moreover, antibody response against T. whipplei is paradoxal. We recently showed that patients with chronic asymptomatic carriage of T. whipplei in fecal specimens had antibodies to T. whipplei; patients with WD did not exhibit or exhibited a low reaction to T. whipplei (29). The lack of antibodies for T. whipplei in patients points to a recent infection in nonimmune persons. The pathogenic role played by T. whipplei in pneumonia is diffi cult to defi ne. In the fi rst patient (specimen no. 5), the role of T. whipplei in pneumonia is probable. In fact, it was the only pathogen we detected using the broadrange PCR-based assay, and it was present at a high level in an immunocompromised patient (5.10 5 copies of the genome) ( Table 1). This patient had community-acquired pneumonia when admitted to the hospital. Results for the 5 other case-patients should be interpreted more cautiously. For the patients from whom specimens no. 10, 82, 86, and 183 were collected, amplifi cation of the 16S rDNA gene showed the presence of several other bacteria usually found in oral fl ora. For the patients from whom specimens 10 and 82 were collected, both of whom had aspiration pneumonia, we hypothesize that T. whipplei was part of the oral or digestive fl ora that contaminated this patient with many other pathogens. It is likely that this was also true for the patients from whom specimens 86 and 183 were collected, both of whom had ventilator-associated pneumonia. For the remaining patient (specimen no. 209), the situation was more complex. In fact, we found oral and dental bacteria, 3 water bacteria, and Pseudomonas stutzeri, which is an environmental bacterium. The role of T. whipplei in this patient remained unclear.
In summary, we reported a series of patients in whom T. whipplei DNA was found in BAL fl uids. In 1 patient (specimen no. 5), the role of T. whipplei in pneumonia is highly convincing. For the 5 other cases, T. whipplei was identifi ed, as were other oral and dental bacteria. The pathogenic role played by T. whipplei in the pneumonia for these 5 patients in conjunction with other bacteria is diffi cult to defi ne but cannot be excluded. Our fi ndings confi rm those reported by Harris et al. (19), who found T. whipplei DNA in the sputum of a child with pneumonia. The role of T. whipplei in ICUs remains to be elucidated. T. whipplei is an ubiquitous bacterium in the digestive tract, and it has been shown that T. whipplei might be present in the saliva of asymptomatic persons (2,30). It is therefore probable that T. whipplei could contribute to the occurrence of aspiration pneumonia along with bacteria present in the oral fl ora. The presence of T. whipplei as the unique identifi ed agent in one of the reported cases is suggestive of a real pathogenicity of this agent.
It is too early to conclude whether T. whipplei is an etiologic agent of aspiration or isolated pneumonia. Nevertheless, the existence of T. whipplei DNA in ≈3% of BAL fl uid samples collected from patients with pneumonia undoubtedly raises questions about its role in the genesis of pneumonia that develops in ICU patients.  Figure. Chest radiograph of patient no. 5, who had communityacquired pneumonia associated with Tropheryma whipplei.