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Seminar

Whipples disease

Thomas Marth, Didier Raoult Whipples disease, or intestinal lipodystrophy, is a systemic infectious disorder affecting mostly middle-aged white men. Patients present with weight loss, arthralgia, diarrhoea, and abdominal pain. The disease is commonly diagnosed by small-bowel biopsy; the appearance of the sample is characterised by inclusions in the lamina propria staining with periodic-acid-Schiff, which represent the causative bacteria. Tropheryma whipplei has been classified as an actinomycete and has been propagated in vitro, which allows the possibility of improving diagnostic strategies, for example through antibody-based detection of the bacillus on duodenal tissue or in circulating monocytes. Cellmediated immunity in active and inactive Whipples disease has subtle defects that might predispose some individuals to symptomatic infection with this bacillus, which probably occurs ubiquitously. Although most patients respond well to empirical antibiotic treatment, some with relapsing disease have a poor outlook. The recent findings and concerted research might allow development of new strategies for diagnosis, treatment, and monitoring of patients with Whipples disease. Infection with Tropheryma whipplei is a good example of the contributions of modern molecular-based techniques to pathogenetic, diagnostic, and therapeutic ideas in clinical medicine. The organism has been cultivated and characterised by molecular-genetic techniques. Clinical manifestation of T whipplei infection seems to occur in the presence of low activity of T-helper cells of type 1 (Th1). These findings, together with other results, have led to an improved pathophysiological understanding of the disease and to new perspectives in treatment strategies. bacterium, antibodies to shigella and streptococcus B were tested; they cross-reacted with the bacteria, allowing preliminary immunohistochemical testing.1518 Several attempts at culture were made but not pursued or reproduced. The Whipples bacillus was tentatively grown in peripheral-blood mononuclear cells deactivated by interleukin 4.19 T whipplei was propagated in human fibroblast cells in 1999 in minimum essential medium with 10% calf serum.20 The first strain has been widely distributed and is now deposited in two official bacterial collections.14 The bacterium grows slowly (estimated doubling time 17 days initially), and has not been grown in axenic (cell-free) media. The site of multiplication is controversial; some researchers believe that the bacterium multiplies in the digestive lumen and is taken up by phagocytosis and slowly degraded in macrophages.7,8 In vitro, the bacteria grow within peripheral-blood mononuclear cells19 and are released from infected cells. Within HeLa cells, T whipplei multiplies actively in an acidic vacuole at pH 5.21 This high acidity impairs the activity of antibiotic compounds22,23 and could well be the reason for the ineffectiveness of many antibiotic regimens. The first attempt to classify the Whipples disease bacterium was by Wilson and colleagues in 1991.24 The investigators sequenced, by universal genomic amplification and PCR, a portion of the 16S rRNA of the presumed bacterium from a duodenal-biopsy sample of a patient with Whipples disease. The sequence was confirmed later, and the name Tropheryma whippelii was

Bacteriology
The cause of Whipples disease remained obscure for years. In his initial description, Whipple reported that silver-stained rod-shaped microorganisms were visible in the vacuole of macrophages, but he did not link this observation with a possible causative agent.1 Later staining procedures with periodic-acid-Schiff (PAS) showed abnormal material.2,3 In 1960 and 1961, observations of bacteria-like bodies were made by transmission electron microscopy, and a bacterial cause was strongly suspected.46 The nature of this bacterium was difficult to establish. It has an atypical morphology for a gram-positive or a gram-negative bacterium.7,8 Many of the observed bacteria in Whipples disease are apparently degraded in macrophages, and intact bacteria can be seen extracellularly.712 The Whipples bacillus does not stain well with gram stain in tissues and is gram-negative in cell culture.10,13,14 The PAS-positive material seen in macrophages and culture is thought to correspond with capsular mucopolysaccharides.5,10 In an effort to stain this
Lancet 2003; 361: 23946
Division of Gastroenterology, Stiftung Deutsche Klinik fr Diagnostik, Wiesbaden, Germany (T Marth MD); and Unit des Rickettsies, CNRS UMR 6020, IFR 48, Facult de Medicine, Marseille, France (Prof D Raoult MD) Correspondence to: Dr Thomas Marth, Division of Gastroenterology, Stiftung Deutsche Klinik fr Diagnostik, Aukammallee 33, 65191 Wiesbaden, Germany (e-mail: marth.gastro2@dkd-wiesbaden.de)

Search strategy and selection criteria

We undertook a computer-aided search of PubMed, which identified 1216 publications on Whipples disease (1140 on Whipple disease). In addition, mostly overlapping, we found 1139 references on intestinal lipodystrophy (111 on Intestinale Lipodystrophie) and six on Morbus Whipple. We had in-depth discussions with participants at the nine partner institutes in the European project on Whipples disease. Finally, we took into account several doctoral theses, several general and specialist book chapters, proceedings, and one monograph to supplement our awareness of the published work.

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Mycobacterium lactis D21343 Mycobacterium liquefaciens X77444 Agromyces ramosus X77447 Curtobacterium citreum X77436 Tropheryma whipplei AF251035 Cellulomonas hominis X82598 Cellulosimicrobium cellulans AB023355 Brevibacterium linens AJ315491 Kocuria rosea Y11330 Arthrobacter globiformis X80736 Micrococcus luteus AJ409096 Arcanobacterium pyogenes X79225 Corynebacterium renale X84249 Pseudonocardia thermophila AJ252830 Mycobacterium leprae X55587 Mycobacterium chelonae AJ419969 Nocardia asteroides Z82231 Rhodococcus equi X80614 Bacillus subtilis AF447803 Clostridium perfringens AB075767
002

Epidemiology and pathogenesis

Whipples disease is rare, and no valid estimate of the incidence is available. The disorder has been described most frequently in white people and in mid-Europe (of 696 cases, 55% were reported from Europe and 38% from North America).40 Only a few cases have been reported in Hispanic, black, Indian, or Asian populations. Many of the published cases come from rural regions, and farmers are frequently among the documented occupations.40,41 The disease sometimes occurs in local clusters.4244 Specific environmental factors or habits have not yet been associated with the disorder. Despite the presumed ubiquitous presence of T whipplei,34 Whipples disease occurs mainly in middleaged individuals (mean age at diagnosis about 50 years) and in about eight times more men than women,40,4547 which supports the effect of genetic factors in the cause. A study by von Herbay and colleagues48 and data from the therapy study SIMW (Study on the Initial Therapy of Morbus Whipple, within the European project on Whipples disease) suggested that the disease is more prevalent in women, but this finding is not confirmed by larger epidemiological series. Several familial cases (brother pairs, father and daughter) have been reported,4951 but most of the analysed cases do not suggest familial components. A genetic susceptibility is suggested by the finding that about 26% of patients (three to four times more than expected) are positive for HLA B27;40,52 however, this characteristic is not found in all populations (for example, Italy53 and Argentina54). The disease can have a chronic relapsing course, and the organism can persist in affected tissues for a long time, even with extended antibiotic therapy. Collectively, these observations suggest that a host factor, putatively of an immunological nature, has a role in the occurence of the disease. Figure 2 shows the defective immune responses seen in patients infected with T whipplei. The presumed immunological defect is likely to be subtle and quite specific for T whipplei, since patients are not generally prediposed to infection with other organisms. Only a few case reports have pointed to the possibility that Whipples disease also occurs in a setting of immunodeficiency55 or immunosuppression56,57 or concomitantly with other infections (eg, in individuals with AIDS,58 nocardiasis, or lambliasis59,60). Results of several immunohistological studies7,61 have shown that despite the influx of macrophages, intestinal tissue has little lymphocytic infiltration and few plasma cells in Whipples disease. Although this lack could reflect a secondary loss of lymphocytes caused by intestinal lymphangiectasia, some investigators have identified more profound phenotypical and functional changes in immunity. Populations of T cells in the lamina propria and the circulation in active Whipples disease are characterised by a low CD4/CD8 T-cell ratio, a shift towards mature T-cell subpopulations (eg, high expression of CD45RO, low CD45RA expression), and increased cell-activation markers.62,63 Reduced proliferative responses of peripheral T cells are found, for example, in response to phytohaemagglutinin, concanavalin A, and antibodies to CD2.52,64 In some cases, as yet unidentified inhibitory serum factors have been identified, which downregulate T-cell-mediated responses.63,64 These changes, and the impaired delayedtype hypersensitivity reaction to recall antigens, are present not only in patients who are acutely ill but also in those with long-standing remission.60,61,63,64 The mucosa contains low numbers of IgA-positive B cells but

Figure 1: Phylogenic position of T whipplei based on 16S rRNA sequencing

The complete nucleotide sequences of the 16S rRNA gene of all tested species were aligned by use of CLUSTAL W.26 Phylogenetic relations among these bacteria were inferred by use of PHYLIP software (version 3.4).27 The distance matrices generated by DNADIST were determined under the assumptions of Kimura and were used to infer dendrograms by the neighbour-joining method.28 Scale bars represent the percentage of nucleotide differences.

suggested (Greek trophe nourishment and eryma barrier, and from Whipple).25 Since culture became available the name was corrected and the organism was officially named Tropheryma whipplei.14 It belongs to the high-G+C phylum of gram-positive bacteria (figure 1). T whipplei is grouped with bacteria mainly found in the environment including cellulomonadacae, but also human-associated bacteria such as Rothia dentocariosa.25,29 T whipplei measures about 0220 m in size.5,7,9 Bacteria of the T whipplei species have some genetic heterogeneity, as shown by sequencing of the 16S23S rDNA interspacer29,30 and the 23S rDNA.31 The genomic variants are associated with the place of residence of the patients and might be geographically distributed.32 T whipplei has a single circular chromosome and small genome size (925 kb); its genome has been sequenced and deposited in Genbank (unpublished). There is no clear link between genotype and symptom pattern; however, the issue of subspecies pathogenic specificity remains unresolved. Some strains could be nonpathogenic, some cause typical Whipples disease, and others cause atypical clinical forms such as infectious endocarditis.12,32,33 The habitat of T whipplei is unknown. Detection of the bacterium by PCR in sewage water in Germany34 and in human faeces12,35 led to speculation that it exists in the environment and contaminates people through drinking water. However, the possibility that human beings are the reservoir cannot be excluded. PCR-based studies have also shown that T whipplei can be amplified from saliva, gastric fluid, and duodenal-biopsy samples of people without Whipples disease.3638 The frequency of samples positive for T whipplei by PCR in people who are asymptomatic depends on the geographical origin of the individual;12 this feature could explain why other researchers did not find such results in other geographical regions.39 The reliability of PCR tests in people without Whipples disease is also controversial.

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Functional effects: cutaneous reactivity to recall antigens Benefit effect of additive therapy with recombinant IFN in antibiotic refractory patients Tropheryma whipplei

IFN

Reduced intracellular degradation of T whipplei Growth of T whipplei in IL 4/IL 10 deactivated monocytes

Macrophage

T cell IL 12

T-cell proliferative response CD4/CD8 ratio Expression of CD11b Expression of mature T cells Expression of naive T cells Th1 responses: IL 2 , IFN Th2 responses: IL 4

Thus, the persistent subtle defect of cellular immunity seems to involve activation and interaction of macrophages and T cells. These processes could result in disturbed phagocytosis and intracellular degradation of T whipplei and allow invasion of the bacillus from the gastrointestinal mucosa to peripheral organs. Future studies with more patients are needed to clarify the exact nature of these defects and possible genetic components.

Clinical features and diagnosis

In vitro production of monocyte-secreted IL 12 and reduced expression of IL 12 in lamina propria

Serum: unidentified inhibitory factors Figure 2: Defective immune responses in T whipplei infection
IL=interleukin. IFN=interferon.

increased numbers of surface-IgM-positive B cells.65 Secretory IgA concentrations measured in intestinal aspirates and humoral immune responses to infectious agents in the periphery are normal.17,66 In addition, serum concentrations of total IgG are normal in most cases, whereas IgM concentrations are low and those of IgA high in acute stages of the disease.43,6668 We have recorded low concentrations of the IgG2 subclass in several patients with Whipples disease, which is produced in response to infection with encapsulated bacteria and is regulated by cell-mediated immune responses and interferon .69 Studies on macrophages in Whipples disease are sparse. Macrophages from infected patients show decreased intracellular degradation70 and a decrease in phagocytosis.71 Patients with either active or inactive Whipples disease have lower than normal numbers of circulating cells expressing CD11b. This molecule serves as a facilitator of microbial phagocytosis, has a role in antigen processing, and mediates intracellular killing of ingested bacteria that is induced by interferon .63 Furthermore, at least during active disease, intestinal macrophages do not express CD11b.72 Thus, a reduction in CD11b expression could indicate a decrease in the ability to cope with intracellular infection. Results of our studies69,73 on 20 patients show that the impaired function of antigen-presenting cells in Whipples disease is related to low production of interleukin 12 in macrophages; this cytokine has important functions in regulation of cellmediated immune responses. Low serum concentrations of interleukin 12p40 have also been recorded (unpublished data). By contrast, functional Th2 responses increase and Th1 responses decrease in peripheral and mucosal T cells73 lending support to the observation that T whipplei replicates in macrophages deactivated by interleukins 4 and 10.19 As a further indication of the pathogenetic relevance of the impaired cellular immune responses, in one patient who had Whipples disease refractory to antibiotic regimens and low concentrations of interferon , treatment with antimicrobials and supplemental recombinant interferon gamma led to clearance of the infection.74

Whipples disease has traditionally been regarded as a gastrointestinal disease, but in most cases, the disease begins insidiously with arthropathy. In one large series,63,75 arthropathy was the first symptom in 63% of patients, preceding the diagnosis of Whipples disease by a mean of 8 years. Arthropathy, in many cases associated with HLA-B27 positivity, consists of chronic, migratory, non-destructive, and seronegative joint disease, mainly in the peripheral joints.40,43 The sacroiliac region is affected in up to a third of patients,76 and radiological changes are found in around a fifth.77 The arthropathy is commonly accompanied by myalgias.40,47,75 Since new diagnostic methods enable detection of T whipplei in synovial fluid,78 which can occur as effusion besides other microscopic signs of synovitis, patients with such disease might be diagnosed earlier in future. Weight loss and diarrhoea are the other major symptoms by the time of diagnosis. Weight loss is found almost invariably. In one large series,43 two-thirds of patients had clinically relevant weight loss (up to 20% of the previous weight) more than 4 years before diagnosis. Gastrointestinal symptoms, which generally begin later and ultimately lead to diagnosis, consist of episodic and watery diarrhoea or steatorrhoea, in many cases accompanied by colicky abdominal pain and, in 2030% of patients, by occult blood in the stool.40,46,47 These symptoms and concomitant anorexia lead to the full picture of a malabsorption syndrome with severe weight loss, weakness, general cachexia, and the associated secondary signs and symptoms. On endoscopy, the lesions of Whipples disease are commonly described as pale yellow shaggy mucosa alternating with an erythematous, erosive, or mildly friable mucosa in the postbulbar region of the duodenum or in the jejunum; alternatively, whitish-yellow plaques can be seen in a patchy distribution.79,80 Therefore, biopsy samples should be taken from both the proximal and distal duodenum or the jejunum. Endoscopy also plays an important part in follow-up. The duodenal mucosa recovers during the first weeks to months of antibiotic treatment, whereas the PAS-positive material in the macrophages can persist for several years; an increase in PAS-positive material after a previous resolution can be the first indicator of a relapse.79,80 A subtype classification of PAS-positive cells indicative of florid or chronic Whipples disease lesions has been suggested, which might be helpful for the clinician in some patients.81 We emphasise that clinical presentation can vary to a great extent owing to differential organ involvement, and some patients present without gastrointestinal manifestations.75,82 Systemic symptoms in about half of

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Approximate frequency Major clinical features Weight loss Arthropathy Diarrhoea Abdominal pain Frequent signs and symptoms Fever Lymphadenopathy Hyperpigmentation Hypotension Peripheral oedema Cardiac murmurs Occult bleeding Myalgia Abdominal mass CNS/eye involvement* Chronic cough Splenomegaly Hepatomegaly Ascites Other signs and symptoms 90% 85% 75% 60% 45% 45% 35% 35% 30% 30% 25% 25% 20% 15% 15% 15% 10% 10% Rare

folds, hepatosplenomegaly, ascites, or other specific organ involvement. MRI is useful in diagnosis of CNS manifestations.93 Laboratory tests can show evidence of malabsorption and protein-losing enteropathy, such as low serum concentrations of carotene, vitamin deficiency (B12, D, K, and folic acid), and low albumin and cholesterol concentrations; additionally, stool fat excretion might be raised, and D-xylose absorption low.40,46,56 Many patients with Whipples disease have, for unknown reasons, pronounced eosinophilia87 and abnormalities of serum immunoglobulins as mentioned in the pathogenesis section.43,61,64 Other non-specific laboratory abnormalities include a high erythrocyte sedimentation rate, increased concentrations of acute-phase proteins such as C-reactive protein, lymphocytopenia, thrombocytosis, and hypochromic anaemia.

Histopathological and laboratory diagnosis

The first diagnostic method is the histological appearance. When the disease is suspected, duodenal-biopsy specimens should be obtained. Depending on the clinical manifestations, other samples should be tested, such as cerebrospinal fluid (CSF), cardiac-valve tissue, lymph nodes, and synovial tissue.46,94 The infiltration of the bowel wall is associated with a widening and flattening of the villi and with dilated lacteals containing yellow lipid deposits, the result of blockade of the villous lymphatics (therefore Whipple suggested the name intestinal lipodystrophy).1,7,11,13 Histological analysis reveals granular foamy macrophages stained purple with PAS (figure 3); in addition, diastaseresistant and silver-positive inclusions representing more or less intact remnants of ingested bacteria might be visible.5,10,13 Duodenal samples from patients with Whipples disease are infiltrated by macrophages; the proportion of macrophages among duodenal cells in these samples can range from under 5% (in the normal host) to 50% (our observation). However, PAS staining is not completely specific; patients with infection caused by Mycobacterium avium-intracellulare, Rhodococcus equi, Bacillus cereus, corynebacterium, histoplasma, or fungi also have PASpositive macrophages (only partly ruled out by a ZiehlNeelsen stain for acid-resistant microorganisms).40,9597 Samples from patients with melanosis coli and histocytosis, and colon samples from patients with Crohns disease, can also be confused in rare instances with Whipples disease.40,98 Involvement of lymphatic tissues, the gastrointestinal tract, and rarely other organs can be accompanied by non-caseating, epithelioid-cell (sarcoid-

*Dementia, ophthalmoplegia, myoclonus, ataxia, nystagmus, visual loss, uveitis, retinitis. eg, pleuritis, pleural effusion, endocarditis, muscle wasting, glossitis, peripheral neuropathy.

Table 1: Signs and symptoms in Whipples disease

patients consist of intermittent, mostly low-grade fever and night sweats. Common features are also peripheral and abdominal lymphadenopathy; the mesenteric lymphadenopathy is identified frequently on radiographs but could also be present as an abdominal mass. Skin hyperpigmentation, particularly affecting light-exposed areas and suggesting Addisons disease (which has not been observed in patients with Whipples disease), has been recorded in up to a third of patients in a large series.41,44 No major organ is excluded from infection by T whipplei, and chronic nonproductive cough or chest pain indicative of lung involvement or pleuritis, polyserositis, ascites, hypotension, and oedema are among other signs and symptoms frequently noted (table 1). Hepatomegaly or splenomegaly can be present in some patients with this disorder. Less frequent involvement has been reported for the genitourinary and endocrine systems.40,4547,56 Cardiac involvement is common and has been reported to be an important clinical sign. It might present as cardiac murmurs, insufficiency of the aortic or mitral valve necessitating replacement, or with the clinical picture of blood-culture-negative endocarditis; many of these cases are diagnosed by histological analysis of the cardiac valves.8388 In many of these patients, endocarditis is isolated; no other evidence of clinical Whipples disease is observed and duodenal biopsy is negative.85,87 A CNS manifestation can first become apparent as a memory disorder, personality change, or dementia in many patients. Other more common clinical signs are ophthalmoplegia, nystagmus, and myoclonia. These are frequently noted in combination with a disturbed sleep pattern, ataxia, seizure, or symptoms of cerebral compression (due to hydrocephalus). Various cranial-nerve symptoms, such as hearing loss and blurred vision, have been reported.40,45,89,90 In some patients, a specific oculomasticatory myorhythmia or myoclonus with ophthalmoplegia has been described.91,92 Such CNS symptoms have a frequency of up to 15% and can occur in rare instances with little or no gastrointestinal involvement.40,92 Radiographic assessment including routine radiographic examination, barium enema, CT, and MRI, often undertaken because of gastrointestinal symptoms, can reveal abdominal lymphomas, a thickening of the mucosal

Figure 3: Histopathology of Whipples disease

(A) Control patient biopsy sample (negative). (B) Whipples disease jejunal biopsy sample. 1=stained by PAS (macrophages are stained in red in patients with Whipples disease). 2=stained by immunohistochemistry with a polyclonal antibody to T whipplei (courtesy of H Lepidi).

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like) granulomas.99 Bacteria can also be reliably visualised by electron microscopy,7,9 but this approach is less convenient. Since Whipples disease is systemic, PASpositive macrophages and electron-microscopically detectable bacilli have been shown in many cell types and in almost all organs.9,13 Immunohistochemistry is of diagnostic help.100102 Nonspecific cross-reactions have prompted very low crossreactions (with shigella and Streptococcus agalactiae) and should be very useful (figure 3). We have shown that immunohistochemistry can detect T whipplei in circulating monocytes of patients with active Whipples disease.101 Culture is currently undertaken only for research purposes in highly specialised laboratories. It may not become a method of diagnosis until culture conditions are improved. T whipplei grows slowly in human fibroblasts (MRC 5 and HEL cells),14 Hela cells, and human peripheral-blood monocytes.19 The major difficulty is that primoculture takes a very long time. Our first successful culture showed a cytopathic effect in cells after 2 months. Antibiotic treatment of patients precludes isolation of the bacterium (unpublished data). Culture from contaminated samples necessitates use of an antibiotic cocktail in the culture medium.100 PCR gene amplification is a promising technique. However, discrepancies occur, depending on the team involved and the samples tested. Positive PCR results were reported with testing of gastric fluid, small-bowel samples, and saliva from patients without disease.3638 These results were not confirmed by other teams.39,102 We reported quantitative detection of T whipplei RpoB sequence by realtime PCR and showed that we can identify a cut-off on the basis of the number of DNA copies to avoid false-positive results.103 We detected 102 to 105 copies in digestive samples in infected patients and none in 150 controls. Quantitative PCR could also help in the future, as suggested by regular PCR,103 to follow up treated patients. Several gene sequences are available94 based on 16S rRNA (such as interspacer), 23S rRNA, or RpoB.104 We recommend before definitive diagnosis, when atypical cases are reported, use of at least two PCR tests based on primers obtained from two different genes to avoid false-positive results caused by contamination. Samples useable for PCR are duodenal-biopsy tissue, synovial fluid, lymph nodes, cardiac valve, vitreous humour, and CSF. The usefulness of saliva and faeces for diagnosis remains unclear.35,36 Blood is not reproducibly a good sample for this purpose.105,106 DNA extraction is a crucial step in the procedure, and several protocols have been proposed, which could be used on paraffin-embedded tissues.91,107,108 There is a risk of contamination with PCR, which is higher with semi-nested or nested methods. Results have to be interpreted with caution and according to the clinical situation. Serology gave promising preliminary results,20 but after subcultures there is an antigenic shift of the bacterium, and crude antigen lacks specificity after a few subcultures in vitro (unpublished data). Biological monitoring of treated patients with Whipples disease has not been established. At present, there is no clear link between any of the tests used for diagnostic purposes and the achievement of remission in patients. PCR on repeated samples is inefficient for prediction of outcome.108 PAS staining generally is not sufficiently predictive in our experience, because PASpositive material clears slowly during treatment. The clinician always has to interpret the histopathological and laboratory findings in view of the clinical presentation of the patientie, treatment should not be started for a positive PCR test without clinical correlation. In cases of doubt, a specialist should be contacted.

Number of patients treated Initial treatment* Tetracycline Penicillin plus streptomycin Trimethoprim-sulfamethoxazole Other antibiotics Total
*From references 97, 112.

Relapses 322% 118% 43% 276% 250%

115 34 23 29 201

Table 2: Frequency of relapse in Whipples disease

Treatment and prognosis

Untreated Whipples disease can be fatal. However, in many patients with the disease, antibiotic therapy leads to rapid improvement in clinical status and to lasting remission.11,40,109111 Diarrhoea and fever can resolve as quickly as within 1 week of the start of therapy, and arthropathy and other symptoms improve in many cases after a few weeks. Clinical improvement is generally accompanied by a normalisation of laboratory findings and gradual reconstitution of the villous architecture of the small intestine. Immunological abnormalities, such as increased IgA or shifts in T-cell subpopulations, resolve within a few months in most patients, but the subtle defect in cell-mediated immunity persists, as mentioned earlier.40,46 In the past, various antibiotic regimens were used on an empirical basis. Many patients were treated up to the 1980s with a 2-week course of intravenous penicillin plus streptomycin followed by oral tetracycline.110 Tetracycline treatment seems to be associated with a high frequency of relapse (table 2), so trimethoprim-sulfamethoxazole (160/800 mg orally twice daily) given for at least a year is now recommended (panel).46,111 In addition, tetracycline does not cross the bloodbrain barrier to a relevant extent, and many patients with Whipples disease have a positive PCR for T whipplei in the CSF or a CNS manifestation.111113 In the case of sulphonamide intolerance, second-line regimens including minocycline, tetracycline, or oral penicillin have been applied, and other antibiotics such as fluoroquinolones and cephalosporins have be used on an individual basis. Oral treatment should be preceded, especially in patients who are severely ill, by a 2-week course of parenteral therapy, which can consist, on the basis of available clinical data, of ceftriaxone (2 g per day intravenously) or treatment with another antibiotic that readily penetrates the blood-brain barrier.53,111,114 In patients who are severely ill, replacement therapy is indicated similar to other malabsorption syndromes. No prospective studies are available on the choice or duration of antibiotic treatment. Culture of T whipplei and

Currently recommended treatment

2 weeks parenteral therapy
Ceftriaxone (or penicillin plus streptomycin)

Long-term therapy (1 year)

Trimethoprim-sulfamethoxazole (or tetracycline or minocycline)

Individual therapeutic approaches on experimental basis

Primary CNS manifestation, relapse with CNS manifestation, antibiotic refractory (two or more relapses), antibiotic-resistant course. Contact: Prof T Marth, Division of Gastroenterology, Stiftung Deutsche Klinik fr Diagnostik, Aukammallee 33, 65191 Wiesbaden, Germany (tel +49 611 577 628; fax + 49 611 577 460; email marth.gastro2@dkd-wiesbaden.de) or Prof G E Feurle, Innere Medizin I, DRK Krankenhaus, D 56566 Neuwied (tel +49 2631 981401)
Note: no prospective therapy trial is available for empirical treatment strategies. Doctors should consider including newly diagnosed and refractory patients into the prospective treatment trial SIMW. Contact the European project on Whipples disease (QLGI-CT-2002-01049). (www.whipplesdisease.info or whipple@dkd-wiesbaden.de).

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development of susceptibility tests should enable definition of more adequate treatment regimens for Whipples disease; prospective trials will be required to allow therapy on the basis of clinical evidence. Thus, we strongly encourage early contact with specialised centres for every newly diagnosed and refractory patient. The inclusion of patients into the first prospective antibiotic trial in Whipples disease (SIMW) is recommended (panel). The study is investigating use of either ceftriaxone or meropenem intravenously for 2 weeks followed by oral trimethoprimsulfamethoxazole for 1 year to prevent CNS manifestations, and the possibility of treating patients refractory to conventional drugs with supportive interferon gamma. A follow-up European trial within the European project on Whipples disease (a consortium of nine institutes, www.whipplesdisease.info) based on data from susceptibility testing will, besides studies on the pathogenesis and diagnosis of the disorder, compare long-term therapy with new substances with trimethoprim-sulfamethoxazole (panel). If the patients have a good clinical response, they can simply be followed up with duodenal biopsies 6 months and 12 months after diagnosis.79 Antibiotic treatment can generally then be stopped if no PAS-positive material is identified. In the rare cases in which bacterial material persists, a more closely followed therapy must be continued, and an alternative antibiotic regimen should be considered. Cerebral manifestations of Whipples disease occur more frequently in a relapse and have a bad prognosis.111 Follow-up of these patients includes analysis of the liquor fluid every 6 months until bacterial material is undetectable.112 The rate of clinical relapses seems to be lower but still significant after treatment with trimethoprim-sulfamethoxazole than with tetracycline therapy.112 Some patients have an antibiotic-refractory disease course and others have a primary or recurrent CNS manifestation for which beneficial treatment still needs to be defined. The new findings in the pathogenesis of Whipples disease on deficient cellular immunity might lead to developments in the therapeutic approach.

Immunohistochemistry of circulating monocytes, or embedded tissues, could facilitate retrospective diagnostic as well as non-invasive procedures. New PCR techniques with higher sensitivity and specificity might be useful to test samples such as faeces and serum. Follow-up of patients with Whipples disease could be based on new tests such as quantitative PCR and immunohistochemistry, which remain to be assessed for this purpose. Other diagnostic methods, such as serology, should be developed. Specific epitopes of the bacterium can be identified by monoclonal antibodies,116 and recombinant proteins selected and used as serological reagents. Finally, identification of the risk factors of the disease, exposure, and host predisposition (ie, immunogenetic host factors that have a role in the clinical manifestation) should help in prevention. The many unanswered questions and the rarity of the disorder necessitate cooperative studies to elucidate improved strategies for diagnosis and treatment of Whipples disease.
Conflict of interest statement
D Raoult has patented the culture process, the serology, and the RpoB sequence of T whippei as a diagnostic procedure. T Marth has no conflict of interest to declare.

Role of the funding source

This work was supported by the Programme Hospitalier de Recherche Clinique, 2001 numero UF1658 (French Ministry of Health). The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or in the writing of the report.

References
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Future perspectives
The reservoir of T whipplei should be identified beyond doubt. At present, environment waste is suspected to be contaminated, and the atypical geographical distribution can be explained by unknown environmental factors. The true prevalence of the infection by T whipplei may differ from that of recognised Whipples disease. Benign forms and atypical manifestations without PAS-positive foamy macrophages could exist. The complete clinical range, including infectious endocarditis, might differ from what we know now. The genome has already been completely sequenced, and the final annotation is on its way (unpublished data). It should provide information about the physiology of the bacterium and many DNA sequences to be used for diagnostic purposes. Such methods could allow control for all atypical results of amplification by a second or third PCR or consensus PCR procedures, increasing the predictive value of the result and could also clarify whether asymptomatic carriers exist. Antibiotic-susceptibility testing could be helpful, because empirical treatment regimens have been disappointing and many relapses occur. The alkalinisation by lysosomotropic agents of the macrophage vacuole in which T whipplei resides could be crucial as in chronic infection with C burnetii; this procedure also restores the bactericidal effect of doxycycline.115

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