Tuberculosis drug resistance in an area of low endemicity in 2004 to 2006: semiquantitative drug susceptibility testing and genotyping.

We determined the quantitative levels and the genetic mechanisms of resistance in drug-resistant clinical isolates of Mycobacterium tuberculosis sampled over a period of 3 years (n = 45; 17 of the isolate were multidrug resistant). Our results led us to hypothesize that some strains categorized as resistant to isoniazid, ethambutol, or streptomycin by standard laboratory procedures of in vitro drug susceptibility testing may still respond to a treatment regimen that includes these agents.

Genotyping reveals a wide heterogeneity of Tropheryma whipplei.

Tropheryma whipplei, the causative agent of Whipple's disease, is associated with various clinical manifestations as well as an asymptomatic carrier status, and it exhibits genetic heterogeneity. However, relationships that may exist between environmental and clinical strains are unknown. Herein, we developed an efficient genotyping system based on four highly variable genomic sequences (HVGSs) selected on the basis of genome comparison. We analysed 39 samples from 39 patients with Whipple's disease and 10 samples from 10 asymptomatic carriers. Twenty-six classic gastrointestinal Whipple's disease associated with additional manifestations, six relapses of classic Whipple's disease (three gastrointestinal and three neurological relapses), and seven isolated infections due to T. whipplei without digestive involvement (five endocarditis, one spondylodiscitis and one neurological infection) were included in the study. We identified 24 HVGS genotypes among 39 T. whipplei DNA samples from the patients and 10 T. whipplei DNA samples from the asymptomatic carriers. No significant correlation between HVGS genotypes and clinical manifestations of Whipple's disease, or asymptomatic carriers, was found for the 49 samples tested. Our observations revealed a high genetic diversity of T. whipplei strains that is apparently independent of geographical distribution and unrelated to bacterial pathogenicity. Genotyping in Whipple's disease may, however, be useful in epidemiological studies.

Cloning and sequencing an unknown gene of Tropheryma whipplei and development of two LightCycler PCR assays.

Whipple's disease is a rare infectious illness that can affect any organ system in the body. It is caused by Tropheryma whipplei, a Gram-positive rod-shaped bacterium with a high G + C content, classified within the actinobacteria. For decades, laboratory detection has been based on microscopy and the periodic acid-Schiff (PAS) staining of biopsies. Recently, PCR has become a useful tool to detect T. whipplei DNA in various clinical specimens. However, a positive PCR result does not confirm Whipple's disease as it has been shown that asymptomatic persons can harbor T. whipplei DNA. Since there is not yet much known about the genome of T. whipplei, genome-walking represents a convenient method to determine unknown gene sequences. Starting from a RAPD fragment we have sequenced and cloned an open reading frame (ORF) of 843 bp. Two real-time PCR assays targeting the ORF fragment and the 16S rRNA gene, respectively, were developed. Compared to a conventional 16S rRNA PCR system the ORF LightCycler assay proved to be very specific (100%) but not sufficiently sensitive (62.4%). In contrast, the 16S rRNA LightCycler assay showed a sensitivity of 95.7% and a specificity of 97.8%. Thus, the 16S rRNA gene assay but not that targeting the new ORF is a suitable alternative to conventional PCR methods.

Detection of Tropheryma whipplei DNA in feces by PCR using a target capture method.

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.