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1.
Evaluation of a specific nested PCR targeting domain III of the 23S rRNA gene of "Tropheryma whippelii" and proposal of a classification system for its molecular variants
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"Tropheryma whippelii"-associated infections are usually confirmed histopathologically by using light microscopy. PCR assays targeting the 16S rRNA gene (16S rDNA) of "T. whippelii" are increasingly being applied for this purpose. Compared to microscopic analysis, PCR seems to be more sensitive, as indicated by the fact that several cases of Whipple's disease with negative histopathological findings but positive PCR results have been reported. Considering the lack of pathognomonic clinical features for this disease and the fact that "T. whippelii" DNA has repeatedly been found in patients without clinical Whipple's disease, such PCR results should be confirmed by additional tests. We have, therefore, evaluated a "T. whippelii"-specific nested PCR targeting domain III of the 23S rDNA with 41 clinical specimens known to contain "T. whippelii" 16S rDNA. All of these specimens were also positive for "T. whippelii" 23S rDNA. The specificity of the test was shown by sequencing of the amplicons and by the absence of amplicons in 38 negative controls. We consider this PCR test to be a suitable tool for confirming the presence of "T. whippelii" DNA in specimens with inconclusive histopathological findings. The information derived from sequencing of the partial "T. whippelii" 23S rDNA was then combined with our recent data of the 16S-23S rDNA spacer region of this organism. Overall, four different rDNA types are recognized in our proposed classification system for molecular variants of "T. whippelii." This preliminary scheme may provide a basis for further epidemiological and clinical studies with "T. whippelii" and associated diseases. 相似文献
2.
Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of "Tropheryma whippelii" and its use for detection of "T. whippelii" in clinical specimens by PCR
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Using broad-spectrum primers, we have amplified, cloned, and sequenced a 620-bp fragment of the "Tropheryma whippelii" heat shock protein 65 gene (hsp65) from the heart valve of a patient with Whipple's endocarditis. The deduced amino acid sequence shows high similarity to those from actinobacteria, confirming that "T. whippelii" is indeed a member of this phylum. Based on the nucleotide sequence, we have developed a "T. whippelii"-specific seminested PCR. Seventeen patients shown to be positive by 16S ribosomal DNA (rDNA) PCR and/or internal transcribed spacer (ITS) PCR were also positive by hsp65 PCR. All 33 control specimens from patients without Whipple's disease and negative for "T. whippelii" by both seminested 16S rDNA and ITS PCR remained negative. All amplicons digested with XhoI revealed an identical restriction pattern. Eight of the 17 hsp65 amplicons representing all three previously described ITS types were sequenced. Three of the amplicons showed slight differences, but none of the mutations detected affected the amino acid sequence of the corresponding protein. We conclude that the hsp65 gene is a suitable target for the specific detection of "T. whippelii." Its product represents a putative antigen for a future serodiagnostic assay. 相似文献
3.
Whipple's disease is a rare multisystemic bacterial infection with variable clinical manifestations. For decades, the laboratory diagnosis was based on the demonstration of periodic acid Schiff-positive inclusions in macrophages of gastrointestinal biopsies. PCR has improved the diagnosis of Whipple's disease due to its increased sensitivity compared to histopathological analysis. To avoid invasive procedures for taking specimens, we have investigated the possibility of detecting Tropheryma whipplei DNA in feces rather than in biopsies or gastric aspirate of patients with and without Whipple's disease. Total bacterial DNA was isolated from stool specimens using Qiagen columns followed by a T. whipplei-specific hybridization step with a biotinylated capture probe and streptavidin-coated magnetic particles. The captured DNA was then amplified using the same seminested PCR targeting the 16S rRNA gene of the organism that had been applied to other specimens without capturing. For five of eight patients with Whipple's disease, duodenal biopsies and stool samples were PCR positive, whereas for the three other patients, both specimens were PCR negative. Of 84 patients without Whipple's disease, 75 tested negative in the duodenal biopsy and in the stool sample. For four, both specimens were positive. Five patients tested positive in the stool sample but not in the biopsy. However, for three of these five patients, the gastric aspirate had been PCR positive, indicating that the stool PCR result was true rather than false positive. Compared to PCR from duodenal biopsies, stool PCR has a sensitivity of 100% and a specificity of 97.3%. Additionally, 15 PCR-positive and 22 PCR-negative stool samples were extracted using the Invisorb Spin Stool DNA kit. The simplified stool extraction showed 93.3% sensitivity and 95.5% specificity compared to the target capture method. We conclude that PCR with stool specimens with either extraction method is a sensitive and specific diagnostic tool for the detection of T. whipplei DNA and one not requiring invasive sampling procedures. 相似文献
4.
Gunia S Behrens MH Stosiek P 《APMIS : acta pathologica, microbiologica, et immunologica Scandinavica》2004,112(3):222-224
Whipple's disease (WD) is a rare disorder caused by the gram-positive actinomycete Tropheryma whippelii (1). An oral route of acquisition is presumed (2). WD is a chronic systemic infection morphologically characterized by foamy PAS-positive macrophages. This case report depicts the diagnosis and clinical management of a woman who presented with atypical cardial manifestations of WD and died from severe cardiac impairment by Tropheryma whippelii which was confirmed by polymerase chain reaction. Histological assessment of myocardial specimens obtained at autopsy showed PAS-positive macrophages accumulating within foci of interstitial myocardial fibrosis. Immunohistochemical staining demonstrated myocarditis with lymphocytes and neutrophilic granulocytes, which has not been reported so far in the course of WD. 相似文献
5.
Michel Drancourt Antoine Carlioz Didier Raoult 《Journal of clinical microbiology》2001,39(7):2425-2430
Until recently no isolate of Tropheryma whippelii was available, and therefore genetic studies were limited to those based on PCR amplification of conserved genes. In this study we determined the nucleotide sequence of rpoB (encoding the beta-subunit of RNA polymerase) from a cultured strain of T. whippelii using degenerate consensus PCR and genome walking. The T. whippelii rpoB consists of 3,657 bp with a 50.4% GC content and encodes 1,218 amino acids with a calculated molecular mass of 138 kDa. Comparison of T. whippelii RpoB with other eubacterial RpoB proteins indicated sequence similarity ranging from 57.19 (Mycoplasma pneumoniae) to 74.63% (Mycobacterium tuberculosis). Phylogenetic analysis of T. whippelii based on comparison of its RpoB sequence with sequences available for other bacteria was consistent with that previously derived from the 16S ribosomal DNA (rDNA) sequence, indicating that it belongs to the actinomyces clade. The sequence comparison allowed the design of a primer pair, TwrpoB.F and TwrpoB.R, specific for T. whippelii rpoB. When incorporated into a PCR, this primer pair allowed the detection of T. whippelii rpoB in three of three 16S rDNA PCR-positive biopsy specimens and zero of seven negative controls. rpoB could therefore be targeted in PCR-mediated detection and identification of this emerging bacterial species. This approach has previously been shown useful for the identification of related mycobacteria. This study underscores that a method involving isolation and then propagation of emerging bacteria is a useful way to quickly achieve extensive molecular knowledge of these pathogens. 相似文献
6.
von Herbay A 《Der Pathologe》2001,22(1):82-88
Since the microbiological discovery of Tropheryma whippelii, Whipple's disease has attracted to new attention in clinical medicine. As small intestinal biopsy is the diagnostic procedure, the impact of knowledge about the histopathological features of Whipple's disease and its differential diagnosis has increased. PAS-positive macrophages in the intestinal mucosa are the diagnostic hallmark, but further subtyping of cells is important. In Whipple's disease macrophages with intensely PAS-positive granular particles in the cytoplasm (type 1) should be distinguished from cells with faintly PAS-positive cytoplasm without granular particles (type 3). The latter type of macrophages may persist even for many years but does not affect a diagnosis of intestinal remission. However, as systemic infection with T. whippelii is common, but intestinal biopsy specimens are not representative for other organs, additional investigations are performed. These include analysis of the cerebrospinal fluid by means of cytology and polymerase chain reaction, even in patients without neurological symptoms. For ascertaining eradication of T. whippelii in the cerebrospinal fluid, polymerase chain reaction is more reliable than cytology. 相似文献
7.
M Maiwald H J Meier-Willersen M Hartmann A von Herbay 《Journal of clinical microbiology》1995,33(5):1354-1356
A case of an AIDS patient infected with the Whipple's disease bacterium, Tropheryma whippelii, is reported. A DNA fragment with sequence specificity for the 16S rRNA gene of the bacterium was detected by PCR in a duodenal biopsy specimen from a 55-year-old male patient with AIDS and diarrhea. The biopsy specimen contained periodic acid-Schiff stain-positive macrophages which did not, however, resemble the sickleform-particle-containing cells characteristic of Whipple's disease. This observation raises two possibilities: either the patient had a coincidence of AIDS and Whipple's disease or Tropheryma whippelii acted as an opportunistic pathogen in this immunodeficient patient. The latter explanation is of interest in light of the ongoing discussion of immunologic abnormalities as predisposing factors for Whipple's disease. 相似文献
8.
E D Weinberg 《Medical hypotheses》2001,57(1):59-60
Because of impairment of microbial iron acquisition ability, some potential pathogens can cause disease only in iron loaded hosts. Tropheryma whippelii, the etiologic agent of Whipple's disease, is a possible example. Whipple's disease is non-contagious, occurs mainly in middle-aged white males, and displays many, but not all, of the complications of hereditary haemochromatosis. Tropheryma whippelii is a gastrointestinal commensal that causes disease in persons who have a Th1-Th2 imbalance. Host susceptibility may be exacerbated by iron loading. Consideration should be given to have patients evaluated for levels of interferon-gamma and interleukin-4 as well as for serum ferritin and transferrin iron saturation. 相似文献
9.
Cultivation of the bacillus of Whipple's disease 总被引:23,自引:0,他引:23
Raoult D Birg ML La Scola B Fournier PE Enea M Lepidi H Roux V Piette JC Vandenesch F Vital-Durand D Marrie TJ 《The New England journal of medicine》2000,342(9):620-625
BACKGROUND: Whipple's disease is a systemic bacterial infection, but to date no isolate of the bacterium has been established in subculture, and no strain of this bacterium has been available for study. METHODS: Using specimens from the aortic [corrected] valve of a patient with endocarditis due to Whipple's disease, we isolated and propagated a bacterium by inoculation in a human fibroblast cell line (HEL) with the use of a shell-vial assay. We tested serum samples from our patient, other patients with Whipple's disease, and control subjects for the presence of antibodies to this bacterium. RESULTS: The bacterium of Whipple's disease was grown successfully in HEL cells, and we established subcultures of the isolate. Indirect immunofluorescence assays showed that the patient's serum reacted specifically against the bacterium. Seven of 9 serum samples from patients with Whipple's disease had IgM antibody titers of 1:50 or more, as compared with 3 of 40 samples from the control subjects (P<0.001). Polyclonal antibodies against the bacterium were generated by inoculation of the microorganism into mice and were used to detect bacteria in the excised cardiac tissue from our patient on immunohistochemical analysis. The 16S ribosomal RNA gene of the cultured bacterium was identical to the sequence for Tropheryma whippelii identified previously in tissue samples from patients with Whipple's disease. The strain we have grown is available in the French National Collection. CONCLUSIONS: We cultivated the bacterium of Whipple's disease, detected specific antibodies in tissue from the source patient, and generated specific antibodies in mice to be used in the immunodetection of the microorganism in tissues. The development of a serologic test for Whipple's disease may now be possible. 相似文献
10.
Identification of the uncultured bacillus of Whipple's disease. 总被引:51,自引:0,他引:51
D A Relman T M Schmidt R P MacDermott S Falkow 《The New England journal of medicine》1992,327(5):293-301
BACKGROUND. Whipple's disease is a systemic disorder known for 85 years to be associated with an uncultured, and therefore unidentified, bacillus. METHODS. We used a molecular genetic approach to identify this organism. The bacterial 16S ribosomal RNA (rRNA) sequence was amplified directly from tissues of five unrelated patients with Whipple's disease by means of the polymerase chain reaction, first with broad-range primers and then with specific primers. We determined and analyzed the nucleotide sequence of the amplification products. RESULTS. A unique 1321-base bacterial 16S rRNA sequence was amplified from duodenal tissue of one patient. This sequence indicated the presence of a previously uncharacterized organism. We then detected this sequence in tissues from all 5 patients with Whipple's disease, but in none of those from 10 patients without the disorder. According to phylogenetic analysis, this bacterium is a gram-positive actinomycete that is not closely related to any known genus. CONCLUSIONS. We have identified the uncultured bacillus associated with Whipple's disease. The phylogenetic relations of this bacterium, its distinct morphologic characteristics, and the unusual features of the disease are sufficient grounds for naming this bacillus Tropheryma whippelii gen. nov. sp. nov. Our findings also provide a basis for a specific diagnostic test for this organism. 相似文献
11.
Fourteen Years of Severe Arthralgia in a Man without Gastrointestinal Symptoms: Atypical Whipple's Disease 总被引:1,自引:0,他引:1
Fabiola Mancini Serena Sbaragli Gilberto Colivicchi Antonio Cassone Alessandra Ciervo 《Journal of clinical microbiology》2009,47(2):492-495
We report here the case of Whipple's disease in a 60-year-old man with severe arthralgia and systemic disorders but without gastrointestinal manifestations. The patient had different clinical diagnoses over a period of 14 years. We identified Tropheryma whipplei by real-time PCR. Molecular typing was also performed by sequencing the 16S-23S rRNA intergenic spacer region and domain III of the 23S rRNA gene. 相似文献
12.
13.
B. Pron C. Poyart E. Abachin T. Fest C. Belanger C. Bonnet P. Capelle J.-F. Bretagne A. Fabianek L. Girard H. Hagège P. Berche 《European journal of clinical microbiology & infectious diseases》1999,18(1):62-65
Amplification of the 16S rRNA gene of Tropheryma whippelii was performed in eight patients with Whipple's disease and 34 control patients to confirm a diagnosis of Whipple's disease
and to monitor the course of disease. Polymerase chain reaction (PCR) tests were positive before treatment in 13 of 15 tissue
samples from Whipple's disease patients (gut 8/8; lymph nodes 2/2; bone marrow 1/2; peripheral blood 2/3), in contrast to
none of 54 tissue samples from controls. PCR tests converted to negative within 4–6 months in six of the Whipple's disease
patients undergoing therapy. These results show that PCR is a reliable and useful tool for diagnosis of Whipple's disease
and for monitoring bacterial elimination during antibiotic therapy. 相似文献
14.
Geissdörfer W Moos V Moter A Loddenkemper C Jansen A Tandler R Morguet AJ Fenollar F Raoult D Bogdan C Schneider T 《Journal of clinical microbiology》2012,50(2):216-222
"Classical" Whipple's disease (cWD) is caused by Tropheryma whipplei and is characterized by arthropathy, weight loss, and diarrhea. T. whipplei infectious endocarditis (TWIE) is rarely reported, either in the context of cWD or as isolated TWIE without signs of systemic infection. The frequency of TWIE is unknown, and systematic studies are lacking. Here, we performed an observational cohort study on the incidence of T. whipplei infection in explanted heart valves in two German university centers. Cardiac valves from 1,135 patients were analyzed for bacterial infection using conventional culture techniques, PCR amplification of the bacterial 16S rRNA gene, and subsequent sequencing. T. whipplei-positive heart valves were confirmed by specific PCR, fluorescence in situ hybridization, immunohistochemistry, histological examination, and culture for T. whipplei. Bacterial endocarditis was diagnosed in 255 patients, with streptococci, staphylococci, and enterococci being the main pathogens. T. whipplei was the fourth most frequent pathogen, found in 16 (6.3%) cases, and clearly outnumbered Bartonella quintana, Coxiella burnetii, and members of the HACEK group (Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae). In this cohort, T. whipplei was the most commonly found pathogen associated with culture-negative infective endocarditis. 相似文献
15.
Whipple disease is a rare, chronic multisystem disease associated with the recently characterized organism Tropheryma whippelii. Extraintestinal manifestation involving the central nervous system, heart, and joints occasionally occurs. Involvement of the abdominal lymph nodes, especially the mesenteric and periaortic nodes, is not uncommon. However, peripheral lymphadenopathy as the sole clinical manifestation of Whipple disease is rare. We describe 2 patients with Whipple disease whose initial manifestation was lymphadenopathy. Lymph nodes from both patients showed infiltration of the sinuses by macrophages containing periodic acid-Schiff-positive, diastase-resistant, sickle-like structures. Electron microscopic evaluation confirmed the presence of rod-like organisms. DNA from each sample was amplified by the polymerase chain reaction using a specific set of oligonucleotide primers developed against the 16S ribosomal RNA coding sequence of T. whippelii. The histopathologic features and differential diagnosis of lipogranulomatous lymphadenopathy secondary to Whipple disease, as well as use of molecular-based assays, are discussed. 相似文献
16.
Whipple's disease revisited 总被引:6,自引:0,他引:6
Whipple's disease has traditionally been considered to be a rare multisystem disorder dominated by malabsorption. The recent identification of the Whipple's disease bacillus has, using polymerase chain reaction based assays, fueled advances in the investigation, diagnosis, and management of this disease. This leader reviews the aetiology, clinical manifestations, investigation, and treatment of Whipple's disease in the light of this new information. 相似文献
17.
Cerebral Whipple's disease. Diagnosis by brain biopsy 总被引:2,自引:0,他引:2
Whipple's disease, a multisystem chronic granulomatous disease treatable by antibiotics, usually presents clinically with gastrointestinal or joint symptoms. Usually, the diagnosis is substantiated by small intestinal biopsy. This shows diastase-resistant periodic-acid-Schiff-(PAS)-positive inclusions in the cytoplasm of macrophages within the lamina propria. By electron microscopy, this PAS-positive material consists of 1.5 X 0.2-mum bacilli and fine fibrillar material within macrophage phagolysosomes. Rarely, Whipple's disease presents clinically as a primary neurologic disease without gastrointestinal symptoms. Because untreated cerebral Whipple's disease usually progresses rapidly to death, it is imperative to establish the diagnosis promptly. This report describes a case of cerebral Whipple's disease without gastrointestinal symptoms that was diagnosed early by light-and electron-microscopic study of brain biopsy material. 相似文献
18.
Zhongxing Liang Bernard La Scola Didier Raoult 《Clinical and diagnostic laboratory immunology》2002,9(1):156-159
Recent isolation of Tropheryma whipplei (formerly Trophyrema whippelii), the agent of Whipple's disease, from the cardiac valve of a patient with Whipple's disease endocarditis now allows the detection of reactive epitopes that could be used in a serological assay. In order to propose an enzyme-linked immunosorbent assay (ELISA) that uses recombinant T. whipplei antigen, we first determined by Western blotting of human, mouse, and rabbit antisera that the common immunodominant epitope is an 84-kDa protein. We then produced 13 monoclonal antibodies (MAbs) against T. whipplei, 12 of which recognize this immunodominant epitope. These MAbs did not react with phylogenetically closely related bacteria or bacteria previously shown to be cross-reactive with T. whipplei, but they did react with two other strains of T. whipplei isolated, one from an ocular sample and the other from a duodenal biopsy specimen. By confocal microscopy, the MAbs allowed detection of T. whipplei within infected fibroblasts. The identification of the 84-kDa antigen with our MAbs will make it possible to develop a diagnostic antigen for use in a diagnostic ELISA for Whipple's disease. 相似文献
19.
Bosman C Boldrini R Borsetti G Morelli S Paglia MG Visca P 《Journal of clinical microbiology》2002,40(8):3104-3106
The case of a 61-year-old woman with Whipple's disease-associated sicca complex is reported. Tropheryma whipplei infection was diagnosed by histological and ultrastructural examination of the jejunal mucosa and sequence analysis of the bacterial 16S ribosomal DNA. The role of vitamin A malabsorption in sicca complex secondary to Whipple's disease is discussed. 相似文献
20.
The bacterial etiology of Whipple's disease is generally accepted. However, the exact identity of the "Whipple bug" has remained elusive. Indeed, the isolation of several types of bacteria from different patients with Whipple's disease has caused some to speculate that Whipple's disease may have polymicrobial etiology. Our light and electron microscopic studies document the presence of bacilliform organisms lying free in the lamina propria of the duodenal mucosa. Intact and partially degraded bacterial organisms were seen in the phagosomes of macrophages. Indirect immunofluorescence studies demonstrated the presence of multiple bacterial antigens in the lamina propria as well as the macrophage granules. This profile of antigens is similar to the profile demonstrated in four other patients in two previous studies. The occurrence of similar profiles of bacterial antigens in the tissues of different patients with Whipple's disease suggests a single microorganism in the etiology of Whipple's disease. Because of the overlapping features of muciphages and Whipple's cells in the rectal biopsy material, the superiority and reliability of proximal small intestinal biopsy in preference to rectal biopsy are re-emphasized. 相似文献