Detection of three different types of ‘Tropheryma whippelii’ directly from clinical specimens by sequencing, single-strand conformation polymorphism (SSCP) analysis and type-specific PCR of their 16S-23S ribosomal intergenic spacer region

Hinrikson, Hans Peter; Dutly, Fabrizio; Nair, Satheesh; Altwegg, Martin

doi:10.1099/00207713-49-4-1701

Cited by 57 publications

(74 citation statements)

References 16 publications

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“…The DNA was extracted using Qiagen (Hilden, Germany) columns (QIAamp tissue kit) as described by the manufacturer. To perform PCR, the 16 to 23S rDNA intergenic spacer region was amplified and sequenced using primers tw3f and tw4f, domain III of the 23S rRNA gene was amplified and sequenced using primers TW23InsF and TW-InsR2, and the rpoB gene was amplified and sequenced using primers TWRPOB.F and TWRPOB.R, as previously described (6,14,16). PCR products were detected by 1% agarose gel electrophoresis analysis and ethidium bromide staining.…”

Section: Methodsmentioning

confidence: 99%

“…In 1991, Wilson et al used broad-range primers to amplify bacterial 16S rDNA directly from infected tissue and then determined that this bacterium belongs to the high-GϩC-content, gram-positive bacteria among the class Actinomycetes (36). To date, six genotypes (numbered 1 to 6) of T. whipplei have been described based on sequence variation within the 16 to 23S ribosomal DNA (rDNA) intergenic spacer, and two genotypes (named A and B) have been described based on the 23S rRNA gene (14)(15)(16)25).…”

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confidence: 99%

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Culture of Tropheryma whipplei from Human Samples: a 3-Year Experience (1999 to 2002)

et al. 2003

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The culture of Tropheryma whipplei, the bacterium responsible for Whipple's disease, has been established only recently. Our objective is to describe, based on our experience, the culture of T. whipplei in HEL cells detected by immunofluorescence staining. Over 3 years, we received 18 samples for T. whipplei culture from 15 patients with Whipple's disease. Ten duodenal biopsy specimens from 10 patients with digestive symptoms were available. Five cardiac valves and three blood samples from five patients with endocarditis were also available. We correlated the results of culture with the type of sample and the culture procedure. Seven isolates were obtained, and three were subsequently established for more than 4 passages. The mean delay for the primary detection was 30 days. The bacterium was isolated more frequently from sterile specimens (5 of 8) than from duodenal biopsy specimens (2 of 10), but the difference (P ‫؍‬ 0.14) was not significant. Decontamination of digestive samples containing colistin, amphotericin B, and cephalotin or ciprofloxacin did not impair the isolation of T. whipplei. The use of vancomycin precludes the primary isolation (7 of 12 versus 0 of 6; P ‫؍‬ 0.08) and the establishment of T. whipplei (3 of 12 versus 0 of 6; P ‫؍‬ 0.5). Omitting samples cultured with vancomycin, the establishment of the strain was significantly higher when antibiotics were prescribed for no more than 7 days (3 of 4 versus 0 of 8; P ‫؍‬ 0.03). Our results demonstrate that samples must be collected within 1 week of an antibiotic regimen's initiation for the successful establishment of the bacterium.

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Section: Methodsmentioning

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Culture of Tropheryma whipplei from Human Samples: a 3-Year Experience (1999 to 2002)

et al. 2003

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“…These taxonomic relationships were recently confirmed by analyses of sequences derived from the actinobacterial insertion in domain III of the 23S rDNA (8) and hsp65 (22). Taxon-specific 16S rDNA primers have since been used to detect the bacterium in patients (28,39) and sewage effluent (17), while sequence analysis of the 16S-23S rDNA spacer was used for the differentiation of strains into three different groups on the basis of their genotypes (9). Two additional genotypes were also subsequently described (18).…”

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rpoB Sequence Analysis of Cultured Tropheryma whippelii

2001

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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 ␤-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.

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“…The diagnosis of Whipple's disease is usually based on the presence of diastase-resistant non-acidfast periodic acid Schiff (PAS)-positive inclusions in macrophages of intestinal biopsies or by electron microscopy showing the trilamellar membrane of T. whipplei (6). Since the sequences of the rRNA genes are known (14,15,22,28), several PCR systems have been developed to identify parts of its 16S ribosomal DNA (rDNA) (2,4,23), of the 16S-23S rDNA internal transcribed spacer (ITS) (12), or of the 23S rDNA (13). These DNA amplification methods are more sensitive than histopathological analysis.…”

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Detection of Tropheryma whipplei DNA in Feces by PCR Using a Target Capture Method

2002

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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.

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Detection of three different types of ‘Tropheryma whippelii’ directly from clinical specimens by sequencing, single-strand conformation polymorphism (SSCP) analysis and type-specific PCR of their 16S-23S ribosomal intergenic spacer region

Cited by 57 publications

References 16 publications

Culture of Tropheryma whipplei from Human Samples: a 3-Year Experience (1999 to 2002)

Culture of Tropheryma whipplei from Human Samples: a 3-Year Experience (1999 to 2002)

rpoB Sequence Analysis of Cultured Tropheryma whippelii

Detection of Tropheryma whipplei DNA in Feces by PCR Using a Target Capture Method

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