Optimization of quantitative polymerase chain reactions for detection and quantification of eight periodontal bacterial pathogens

BACKGROUND: The aim of this study was to optimize quantitative (real-time) polymerase chain reaction (qPCR) assays for 8 major periodontal pathogens, i.e. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micros, Porphyromonas gingivalis, Prevotella intermedia, Tanerella forsythia and Treponema denticola, and of the caries pathogen Streptococcus mutans. RESULTS: Eighteen different primer pairs were analyzed in silico regarding specificity (using BLAST analysis) and the presence of secondary structures at primer binding sites (using mFOLD). The most specific and efficiently binding primer pairs, according to these analyses, were selected for qPCR-analysis to determine amplification efficiency, limit of quantification and intra-run reproducibility. For the selected primer pairs, one for each species, the specificity was confirmed by assessing amplification of DNA extracts from isolates of closely related species. For these primer pairs, the intercycler portability was evaluated on 3 different thermal cyclers (the Applied Biosystems 7300, the Bio-Rad iQ5 and the Roche Light Cycler 480). For all assays on the different cyclers, a good correlation of the standard series was obtained (i.e. r2 >= 0.98), but quantification limits varied among cyclers. The overall best quantification limit was obtained by using a 2 mul sample in a final volume of 10 mul on the Light Cycler 480. CONCLUSIONS: In conclusion, the proposed assays allow to quantify the bacterial loads of S. mutans, 6 periodontal pathogenic species and the genus Fusobacterium.This can be of use in assessing periodontal risk, determination of the optimal periodontal therapy and evaluation of this treatment

Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women

Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim broth (Todd-Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS.status: publishe

Microflora of the penile skin-lined neovagina of transsexual women

<p>Abstract</p> <p>Background</p> <p>The microflora of the penile skin-lined neovagina in male-to-female transsexuals is a recently created microbial niche which thus far has been characterized only to a very limited extent. Yet the knowledge of this microflora can be considered as essential to the follow-up of transsexual women. The primary objective of this study was to map the neo-vaginal microflora in a group of 50 transsexual women for whom a neovagina was constructed by means of the inverted penile skin flap technique. Secondary objectives were to describe possible correlations of this microflora with multiple patients' characteristics, such as sexual orientation, the incidence of vaginal irritation and malodorous vaginal discharge.</p> <p>Results</p> <p>Based on Gram stain the majority of smears revealed a mixed microflora that had some similarity with bacterial vaginosis (BV) microflora and that contained various amounts of cocci, polymorphous Gram-negative and Gram-positive rods, often with fusiform and comma-shaped rods, and sometimes even with spirochetes. <it>Candida </it>cells were not seen in any of the smears.</p> <p>On average 8.6 species were cultured per woman. The species most often found were: <it>Staphylococcus epidermidis</it>, <it>Streptococcus anginosus </it>group spp., <it>Enterococcus faecalis, Corynebacterium </it>sp., <it>Mobiluncus curtisii </it>and <it>Bacteroides ureolyticus</it>. Lactobacilli were found in only one of 30 women</p> <p>There was no correlation between dilatation habits, having coitus, rinsing habits and malodorous vaginal discharge on the one hand and the presence of a particular species on the other. There was however a highly significant correlation between the presence of <it>E. faecalis </it>on the one hand and sexual orientation and coitus on the other (p = 0.003 and p = 0.027 respectively).</p> <p>Respectively 82%, 58% and 30% of the samples showed an amplicon after amplification with <it>M. curtisii</it>, <it>Atopobium vaginae </it>and <it>Gardnerella vaginalis </it>primer sets.</p> <p>Conclusion</p> <p>Our study is the first to describe the microflora of the penile skin-lined neovagina of transsexual women. It reveals a mixed microflora of aerobe and anaerobe species usually found either on the skin, in the intestinal microflora or in a BV microflora.</p

The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniae and comparison with four other species specific PCR assays

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a challenge.</p> <p>Methods</p> <p>This report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of <it>S. pneumoniae</it>, and its performance compared to other genotypic and phenotypic tests.</p> <p>Results</p> <p>The new PCR assay designed in this study, proved to be specific at 57°C for <it>S. pneumoniae</it>, not amplifying <it>S. pseudopneumoniae </it>or any other streptococcal strain or any strains from other upper airway pathogenic species. PCR assays (psaA, LytA, ply, spn9802-PCR) were previously described for the specific amplification of <it>S. pneumoniae</it>, but <it>psaA</it>-PCR was the only one found not to cross-react with <it>S. pseudopneumoniae</it>.</p> <p>Conclusion</p> <p>Spne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify <it>S. pseudopneumoniae </it>as <it>S. pneumoniae</it>. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.</p

Genotyping of Streptococcus agalactiae (group B streptococci) isolated from vaginal and rectal swabs of women at 35-37 weeks of pregnancy

<p>Abstract</p> <p>Background</p> <p>Group B streptococci (GBS), or <it>Streptococcus agalactiae</it>, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Here we compared different culture media for GBS detection and we compared the occurrence of different genotypes and serotypes of GBS isolates from the vagina and rectum.</p> <p>Methods</p> <p><it>Streptococcus agalactiae </it>was cultured separately from both rectum and vagina, for a total of 150 pregnant women, i) directly onto Columbia CNA agar, or indirectly onto ii) Granada agar resp. iii) Columbia CNA agar, after overnight incubation in Lim broth.</p> <p>Results</p> <p>Thirty six women (24%) were colonized by GBS. Of these, 19 harbored GBS in both rectum and vagina, 9 only in the vagina and 8 exclusively in the rectum. The combination of Lim broth and subculture on Granada agar was the only culture method that detected all GBS positive women. Using RAPD-analysis, a total of 66 genotypes could be established among the 118 isolates from 32 women for which fingerprinting was carried out. Up to 4 different genotypes in total (rectal + vaginal) were found for 4 women, one woman carried 3 different genotypes vaginally and 14 women carried two 2 different genotypes vaginally. Only two subjects were found to carry strains with the same genotype, although the serotype of both of these strains was different.</p> <p>Eighteen of the 19 subjects with GBS at both sites had at least one vaginal and one rectal isolate with the same genotype.</p> <p>We report the presence of two to four different genotypes in 22 (61%) of the 36 GBS positive women and the presence of identical genotypes in both sites for all women but one.</p> <p>Conclusion</p> <p>The combination of Lim broth and subculture on Granada medium provide high sensitivity for GBS detection from vaginal and rectal swabs from pregnant women. We established a higher genotypic diversity per individual than other studies, with up to four different genotypes among a maximum of 6 isolates per individual picked. Still, 18 of the 19 women with GBS from both rectum and vagina had at least one isolate from each sampling site with the same genotype.</p

Identification and genotyping of bacteria from paired vaginal and rectal samples from pregnant women indicates similarity between vaginal and rectal microflora

Background: The vaginal microflora is important for maintaining vaginal health and preventing infections of the reproductive tract. The rectum has been suggested as the major source for the colonisation of the vaginal econiche. Methods: To establish whether the rectum can serve as a possible bacterial reservoir for colonisation of the vaginal econiche, we cultured vaginal and rectal specimens from pregnant women at 35-37 weeks of gestation, identified the isolates to the species level with tRNA intergenic length polymorphism analysis (tDNA-PCR) and genotyped the isolates for those subjects from which the same species was isolated simultaneously vaginally and rectally, by RAPD-analysis. One vaginal and one rectal swab were collected from a total of each of 132 pregnant women at 35-37 weeks of gestation. Swabs were cultured on Columbia CNA agar and MRS agar. For each subject 4 colonies were selected for each of both sites, i.e. 8 colonies in total. Results: Among the 844 isolates that could be identified by tDNA-PCR, a total of 63 bacterial species were present, 9 (14%) only vaginally, 26 (41%) only rectally, and 28 (44%) in both vagina and rectum. A total of 121 (91.6%) of 132 vaginal samples and 51 (38.6%) of 132 rectal samples were positive for lactobacilli. L. crispatus was the most frequently isolated Lactobacillus species from the vagina (40% of the subjects were positive), followed by L. jensenii (32%), L. gasseri (30%) and L. iners (11%). L. gasseri was the most frequently isolated Lactobacillus species from the rectum (15%), followed by L. jensenii (12%), L. crispatus (11%) and L. iners (2%). A total of 47 pregnant women carried the same species vaginally and rectally. This resulted in 50 vaginal/rectal pairs of the same species, for a total of eight different species. For 34 of the 50 species pairs (68%), isolates with the same genotype were present vaginally and rectally and a high level of genotypic diversity within species per subject was also established. Conclusion: It can be concluded that there is a certain degree of correspondence between the vaginal and rectal microflora, not only with regard to species composition but also with regard to strain identity between vaginal and rectal isolates. These results support the hypothesis that the rectal microflora serves as a reservoir for colonisation of the vaginal econiche

Optimal Control Based Eco-Driving: Theoretical Approach and Practical Applications (Eco-rijden gebaseerd op optimale controle: theoretische aanpak en praktische toepassingen)

Eco-driving is adopting an eco-conscious driving style with the potential to reduce fuel consumption in an easy and cost-effective way. This dissertation develops a methodology to calculate the most fuel-efficient driving behavior. Eco-driving is treated as an optimal control problem. Different low-degree quasistatic polynomial fuel consumption models and optimal control methods are evaluated. The proposed methodology uses quadratic fuel consumption models and Pontryagin s maximum principle. An explicit and comprehensive expression for the optimal engine control is derived. Gear shifting is introduced by maximizing or minimizing an additional variable. Simulations reveal some interesting properties of optimal driving behavior. These results are used to propose refined eco-drive guidelines.status: publishe

Brandstof besparen met intelligente software

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Analyis of Eco-Driving Using Polynomial Fuel Consumption Models

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Minimum-fuel control of combustion engine powertrains

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