Showing papers by "Paul H. M. Savelkoul published in 2004"

New developments in the diagnosis of bloodstream infections

Abstract: New techniques have emerged for the detection of bacteria in blood, because the blood culture as gold standard is slow and insufficiently sensitive when the patient has previously received antibiotics or in the presence of fastidious organisms. DNA-based techniques, hybridisation probes, and PCR-based detection or protein-based detection by mass spectroscopy are aimed at rapid identification of bacteria and provide results within 2 h after the first signal of growth in conventional blood cultures. Also, detection of microorganisms directly in blood by pathogen-specific or broad-range PCR assays (eubacterial or panfungal) shows promising results. Interpretation is complex, however, because of detection of DNA rather than living pathogens, the risk of interfering contamination, the presence of background DNA in blood, and the lack of a gold standard. As these techniques are emerging, clinical value and cost-effectiveness have to be assessed. Nevertheless, molecular assays are expected eventually to replace the current conventional microbiological techniques for detection of bloodstream infections.

Detection of bacterial DNA in blood samples from febrile patients: underestimated infection or emerging contamination?

Abstract: We applied real-time broad-range polymerase chain reaction (PCR) to detect bacteraemia in blood from febrile patients. Interpretation of amplification results in relation to clinical data and blood culture outcome was complex, although the reproducibility of the PCR results was good. Sequencing analysis of the PCR products revealed the presence of Burkholderia species DNA while no Burkholderia species grew in culture. The source of this contamination was shown to be the commercial DNA isolation kit used in the automated MagNA Pure Isolation Robot. A high degree of suspicion is required when uncommon or unexpected pathogens are diagnosed by molecular methods as clinical consequences can be serious.

Detection of Helicobacter species DNA by quantitative PCR in the gastrointestinal tract of healthy individuals and of patients with inflammatory bowel disease

Abstract: In many animal species different intestinal Helicobacter species have been described and a few species are associated with intestinal infection. In humans, the only member of the Helicobacter family which is well described in literature is Helicobacter pylori. No other Helicobacter-associated diseases have definitely been shown in humans. We developed a sensitive quantitative PCR to investigate whether Helicobacter species DNA can be detected in the human gastrointestinal tract. We tested gastric biopsies (including biopsies from H. pylori positive persons), intestinal mucosal biopsies and fecal samples from healthy persons, and intestinal mucosal biopsies from patients with inflammatory bowel disease (IBD) for the presence of Helicobacter species. All gastric biopsies, positive for H. pylori by culture, were also positive in our newly developed PCR. No Helicobacter species were found in the mucosal biopsies from patients with IBD (n=56) nor from healthy controls (n=25). All fecal samples were negative. Our study suggests that Helicobacter species, other than H. pylori, are not present in the normal human gastrointestinal flora and our results do not support a role of Helicobacter species in IBD.

Considerations in Evaluating the Applicability of Universal Detection of Oral Pathogens

Abstract: Akihiro Yoshida et al. (4). recently published an interesting paper describing the development of a 5′ fluorogenic nuclease-based real-time PCR assay for quantitative detection of the oral pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The authors provide the description of a set of primers and probe for amplification and detection of a broad range of bacteria based on highly conserved regions in the 16S rRNA gene. Alignment of the forward and reverse primers and probe sequences, however, showed mismatches at the 5′ as well as the 3′ ends of the 16S rRNA sequences with different oral pathogens (Fig. ​(Fig.1).1). Most likely these mismatches interfere with the amplification of P. gingivalis. In addition, according to Yoshida et al., the primers and probe sequences were based on earlier published data (2). We noticed that except for the probe sequence, the primer sequences presented in the publication of Greisen et al. (2) are located 172 and 254 bases, respectively, upstream and 180 bases downstream from the site indicated by Yoshida et al. FIG. 1. Alignment of the 16S rRNA sequence region described by Yoshida et al., including six oral bacteria compared to the E. coli sequence. The locations of the universal primers (Uni152-F and Uni220-R) and the probe (Uni177-T) are identical to the E. coli sequence ... We have tested this universal probe and primer set with one nonoral bacterium and six oral bacteria: Escherichia coli, P. gingivalis, A. actinomycetemcomitans, Tannerella forsythensis (formerly Bacteroides forsythus), Micromonas micros (formerly Peptostreptococcus), Prevotella intermedia, and Fusobacterium nucleatum. Growth of the bacteria, preparation of serial dilutions of pure cultures, DNA isolation, and real-time PCR amplification were performed as described previously (1). The E. coli amplification signals consistently showed an increasing CT range (i.e., decreasing amplification signal) between 15.9 and 26.3, which corresponded to 4.95 × 107 to 4.95 × 104 CFU equivalents/PCR. All the oral bacteria, however, showed a CT range between 29.5 and 33.5, which is at the level of the negative control signal (CT = 29.5). For A. actinomycetemcomitans, an amplification signal was obtained only with a high DNA concentration, corresponding to 10,000 CFU/ml, and the signals obtained with lower DNA concentrations were at the level of the negative control. To validate the DNA isolation, a real-time PCR with a specific primer-probe combination for E. coli (3) and P. gingivalis (1) was used. The amplification signals obtained with the specific PCR on the same DNA confirmed that the amount of DNA present in the dilution corresponded to the amount isolated. In summary, the results with the eubacterium real-time PCR confirm the mismatches in the alignment but are in contradiction with the results on the same strains presented by Yoshida et al. In our laboratory, the primer and probe set described by Yoshida is not universal, does not amplify P. gingivalis, T. forsythensis, M. micros, P. intermedia, or F. nucleatum, and amplifies A. actinomycetemcomitans only at a high DNA concentration. We conclude that the universal real-time probe and primer set published by Yoshida et al. to detect A. actinomycetemcomitans and P. gingivalis does not seem applicable for sensitive detection of oral bacterium species belonging to the major bacterial periodontal pathogens.