Helicobacter species are not detectable by 16S rDNA PCR in bile from Dutch patients with common bile duct stones.

BACKGROUND: Some Helicobacter species colonize the intestinal tract. To explore the possible relation between Helicobacter spp. and gallbladder disorders, we have investigated their presence in bile of patients with biliary obstruction and dilatation of the bile ducts. METHODS: Bile was sampled from 31 Dutch patients with biliary obstruction identified by jaundice and dilatation of the bile ducts on ultrasound. Samples (n = 31) were obtained immediately following cannulation of the common bile duct (CBD) by endoscopic retrograde cholangiopancreatography (ERCP) (n = 29) or by peri-operative puncture of the gallbladder (n = 2). DNA was isolated from bile by binding to diatoms. Helicobacter spp. were detected by a sensitive (detection limit 1 CFU per reaction tube) 16S rDNA PCR with genus-specific primers. Duplicate samples were spiked with Helicobacter pylori DNA and subjected to PCR in order to check for inhibition. RESULTS: 28 patients had CBD stones (bile collected by ERCP (n = 26) or operatively (n = 2)), 2 had a pancreatic head tumor, and in 1 no abnormalities were found. In 1 of 21 amplifiable bile samples (10/31 inhibited) from Dutch patients with CBD stones, H. pylori 16S rDNA was found. CONCLUSION: Our data indicate that CBD stones in Dutch patients are not associated with the presence of Helicobacter spp. in bil

The additional value of real-time PCR in the quantitative detection of periodontal pathogens

Background and Aim: For the analysis of subgingival plaque, anaerobic bacterial culture has been the gold standard for many years. Currently, molecular microbial techniques have become available to identify and quantify target organisms with high specificity and sensitivity. The technique of real-time polymerase chain reaction (RT-PCR) provides a new tool to detect oral pathogens both in oral and non-oral human infections. The aim of this study was to compare the RT-PCR and anaerobic culture for detection and quantification of six periodontal pathogens in periodontal health and disease. Material and Methods: Subgingival plaque samples from 259 adult patients with periodontitis and 111 healthy controls were analysed with quantitative anaerobic culture and quantitative RT-PCR for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Micromonas micros and Fusobacterium spp. Results: All species were more frequently isolated from patients than controls with both culture and RT-PCR. P. gingivalis, T. forsythia and M. micros appeared significant markers for disease with both techniques. P. intermedia was significantly associated with periodontitis by RT-PCR only (OR 9.7), whereas A. actinomycetemcomitans showed a significant relationship by culture only. The critical differences between culture and RT-PCR were culture-negative/PCR-positive samples which amounted to 7% for A. actinomycetemcomitans, 3% for P. gingivalis, 7% for T. forsythia, 20% for P. intermedia, 6% for M. micros, and 0.8% for Fusobacterium spp. in periodontitis patients and 12%, 3%, 2%, 35%, 14% and 0%, respectively, in the periodontally healthy group. Furthermore, periodontitis individuals had significantly higher amount of all of the test species in the subgingival plaque samples compared with healthy subjects. Conclusion: RT-PCR provides a new rapid diagnostic tool and opens the opportunity to detect small numbers of oral pathogens in clinical specimens, which are under the detection limit by culture technique