Phosphorus recovery as struvite from digested sludge - experience from the full scale.

There is an ongoing paradigm shift; wastewater is often not considered a waste any more, but a source of valuable resources: nutrients (N: nitrogen, P: phosphorus, and K: potassium), energy and water. The recovery of phosphorus from municipal wastewater has gained a lot of attention because of limited phosphate rock reserves and associated geopolitics, and pollution of phosphate rock. At the WWTP of Leuven, Aquafin operates a full scale installation to recover phosphorus as struvite from digested sludge. This paper discusses the performance of the struvite plant, pollutants in the struvite, struvite use, and economics.

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

BACKGROUND: Streptococcus pneumoniae is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a challenge. METHODS: This report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of S. pneumoniae, and its performance compared to other genotypic and phenotypic tests. RESULTS: The new PCR assay designed in this study, proved to be specific at 57 degrees C for S. pneumoniae, not amplifying S. pseudopneumoniae 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 S. pneumoniae, but psaA-PCR was the only one found not to cross-react with S. pseudopneumoniae. CONCLUSION: Spne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify S. pseudopneumoniae as S. pneumoniae. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.

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

BACKGROUND: 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. METHODS: A total of 300 swabs was taken from 100 pregnant women at 35-37 weeks of gestation. For each subject, one rectovaginal, one vaginal and one rectal ESwab were collected. Plating onto Columbia CNA agar (CNA), group B streptococcus differential agar (GBSDA) (Granada Medium) and chromID Strepto B agar (CA), with and without Lim broth enrichment, were compared. The isolates were confirmed as S. agalactiae using the CAMP test on blood agar and by molecular identification with tDNA-PCR or by 16S rRNA gene sequence determination. RESULTS: The overall GBS colonization rate was 22%. GBS positivity for rectovaginal sampling (100%) was significantly higher than detection on the basis of vaginal sampling (50%), but not significantly higher than for rectal sampling (82%). Direct plating of the rectovaginal swab on CNA, GBSDA and CA resulted in detection of 59, 91 and 95% of the carriers, respectively, whereas subculturing of Lim broth yielded 77, 95 and 100% positivity, respectively. Lim broth enrichment enabled the detection of only one additional GBS positive subject. There was no significant difference between GBSDA and CA, whereas both were more sensitive than CNA. Direct culture onto GBSDA or CA (91 and 95%) detected more carriers than Lim broth enrichment and subculture onto CNA (77%). One false negative isolate was observed on GBSDA, and three false positives on CA. CONCLUSIONS: In conclusion, rectovaginal sampling increased the number GBS positive women detected, compared to vaginal and/or rectal sampling. Direct plating on CA and/or GBSDA provided rapid detection of GBS that was at least as sensitive and specific as the CDC recommended method of Lim broth subcultured onto non chromogenic agar.

Microflora of the penile skin-lined neovagina of transsexual women.

BACKGROUND: 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. RESULTS: 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. Candida cells were not seen in any of the smears. On average 8.6 species were cultured per woman. The species most often found were: Staphylococcus epidermidis, Streptococcus anginosus group spp., Enterococcus faecalis, Corynebacterium sp., Mobiluncus curtisii and Bacteroides ureolyticus. Lactobacilli were found in only one of 30 women. 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 E. faecalis on the one hand and sexual orientation and coitus on the other (p = 0.003 and p = 0.027 respectively). Respectively 82%, 58% and 30% of the samples showed an amplicon after amplification with M. curtisii, Atopobium vaginae and Gardnerella vaginalis primer sets. CONCLUSION: 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.

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

BACKGROUND: Group B streptococci (GBS), or Streptococcus agalactiae, 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. METHODS: Streptococcus agalactiae 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. RESULTS: 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.Eighteen of the 19 subjects with GBS at both sites had at least one vaginal and one rectal isolate with the same genotype.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. CONCLUSION: 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.

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.

High-rate activated sludge systems combined with dissolved air flotation enable effective organics removal and recovery.

High-rate activated sludge (HRAS) systems typically generate diluted sludge which requires further thickening prior to anaerobic digestion (AD), besides the need to add considerable coagulant and flocculant for the solids separation. As an alternative to conventional gravitational settling, a dissolved air flotation (DAF) unit was coupled to a HRAS system or a high-rate contact stabilization (HiCS) system. The HRAS-DAF system allowed up to 78% removal of the influent solids, and the HiCS-DAF 67%. Both were within the range of values typically obtained for HRAS-settler systems, albeit at a lower chemical requirement. The separated sludge had a high concentration of up to 47 g COD L-1, suppressing the need of further thickening before AD. Methanation tests showed a biogas yield of up to 68% on a COD basis. The use of a DAF separation system can thus enable direct organics removal at high sludge concentration and with low chemical needs.

Fetal and infant health outcomes among immigrant mothers in Flanders, Belgium.

OBJECTIVE: To compare fetal and infant mortality between immigrant and native-born mothers in Flanders, Belgium. METHODS: In a population-based study, data from 326 166 neonatal deliveries, collected by the Study Center for Perinatal Epidemiology and the Belgian Civil Birth Registration system between January 2004 and December 2008, were analyzed. Immigrant mothers were defined as women born in any country other than Belgium, and were grouped by country of origin according to the World Bank Atlas definition of low-, middle-, and high-income countries. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated to evaluate the association between immigration and fetal/infant outcome. RESULTS: In univariate analysis, fetal and infant mortality rates were significantly higher among immigrants than among native-born mothers (fetal: crude OR, 1.50; 95% CI, 1.29-1.75; infant: crude OR, 1.47; 95% CI, 1.29-1.67). Fetal/infant death rates were highest among mothers originating from low-income countries. In multivariate analysis, however, most differences became non-significant: only the early neonatal death rate remained significantly higher (adjusted OR, 1.30; 95% CI, 1.06-1.60), whereas the fetal death rate appeared lower (adjusted OR, 0.67; 95% CI, 0.57-0.80), among immigrant mothers. CONCLUSION: After adjustment for relevant characteristics, fetal/infant mortality was comparable between immigrant women and native-born women in Flanders.

Elevated soluble triggering receptor expressed on myeloid cells (sTREM)-1 levels in maternal serum during term and preterm labor.

BACKGROUND: Infection and inflammation are important mechanisms leading to preterm birth. Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) belongs to a family of cell surface receptors that seems to play an important role in fine-tuning the immune response. It has been demonstrated that sTREM-1 is involved in bacterial infection as well as in non-infectious inflammatory conditions. Few studies have investigated serum sTREM-1 expression during preterm labor. Therefore, the purpose of this study was to assess sTREM-1 concentrations in maternal serum during term and preterm labor. METHODS: This case control study included 176 singleton pregnancies in the following groups: patients in (1) preterm labor, delivered before 34 weeks (PTB) (n = 52); (2) GA matched controls, not in labor, matched for gestational age (GA) with the PTB group (n = 52); (3) at term in labor (n = 40) and (4) at term not in labor (n = 32). sTREM-1 concentrations were determined by enzyme-linked immunoassay. RESULTS: sTREM-1 was detected in all serum samples. Median sTREM-1 concentrations were significantly higher in women with PTB vs. GA matched controls (367 pg/ml, interquartile range (IQR) 304-483 vs. 273 pg/ml, IQR 208-334; P<0.001) and in women at term in labor vs. at term not in labor (300 pg/ml, IQR 239-353 vs. 228 pg/ml, IQR 174-285; P<0.001). Women with PTB had significantly higher levels of sTREM-1 compared to women at term in labor (P = 0.004). Multiple regression analysis, with groups recoded as three key covariates (labor, preterm and rupture of the membranes), showed significantly higher sTREM-1 concentrations for labor (+30%, P<0.001) and preterm (+15%, P = 0.005) after adjusting for educational level, history of PTB and sample age. CONCLUSIONS: sTREM-1 concentrations in maternal serum were elevated during spontaneous term and preterm labor and sTREM-1 levels were significantly higher in preterm labor.

Rapid and accurate identification of isolates of Candida species by melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA).

Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA) as an identification tool for distinguishing between 16 Candida spp., i.e. Candida albicans, Candida bracarensis, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lipolytica, Candida lusitaniae, Candida nivariensis, Candida norvegensis, Candida parapsilosis, Candida tropicalis and Candida sojae, and Saccharomyces cerevisiae and one species pair, i.e. Candida metapsilosis/Candida orthopsilosis. Starting from a cultured isolate, ITS2-MCA led to differentiation of these species within 6 h. According to our findings, ITS2-MCA offers a simple, rapid and cost-effective method for identification of cultured isolates of the clinically most relevant and prevalent Candida species. Further studies will be necessary to evaluate how it performs on mixed samples and clinical samples.

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 µl sample in a final volume of 10 µl 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.

Strong correspondence in bacterial loads between the vagina and rectum of pregnant women.

We sampled the vagina and rectum in 71 pregnant women and bacterial loads of Lactobacillus crispatus, L. jensenii, L. gasseri, L. iners, Gardnerella vaginalis and Atopobium vaginae were determined by culture and quantitative PCR (qPCR). Culture and qPCR results differed substantially with regard to the evaluation of vaginal and rectal occurrence of the six species tested. The vaginal-rectal prevalence of L. crispatus, L. jensenii, L. gasseri, L. iners, G. vaginalis and A. vaginae as established by culture vs. PCR was 32.3 vs. 91.5%, 32.3 vs. 77.4%, 28.1 vs. 91.5%, 12.6 vs. 68.5%, 12.6 vs. 74.6% and 5.6 vs. 69.0%, respectively. Using qPCR, a significant positive correlation was found between vaginal and rectal loads of L. crispatus (p < 0.0001), L. jensenii (p < 0.0001), L. gasseri (p = 0.005), L. iners (p = 0.003) and A. vaginae (p = 0.002). In summary, significant correlations between quantities of vaginal and rectal lactobacilli and of Atopobium vaginae were established by means of qPCR, indicating strong correspondence of vaginal and rectal microflora, not only in the occurrence of certain species in both niches, but also of cell densities per bacterial species.

In vivo evaluation of the vaginal distribution and retention of a multi-particulate pellet formulation.

Non-disintegrating microcrystalline cellulose pellets (MCC) and disintegrating starch-based pellets were evaluated as new vaginal drug delivery forms and compared with a powder formulation. Pellets and powder were packed in a HPMC or hard gelatine capsule and vaginally administered to five series of five healthy volunteers. Distribution and retention of the multi-particulate formulation was monitored by colposcopy and swabbing. Capsule disintegration in the vagina was slow. MCC pellets clustered around the fornix 3h after administration, and after 24h only a few pellets were detected in the vaginal cavity. In contrast, starch-based pellets already started to disintegrate 6h after administration, resulting in a complete coverage of the vaginal mucosa after 24h in 8 out of 10 volunteers. The powder formulation had a better distribution after 6h, although after 24h almost no powder remained in the vagina. These results were confirmed by swabbing to determine the amount of riboflavin sodium phosphate (used as marker) distributed in the different vaginal regions.

A pilot study evaluating the safety of vaginal administration of a multi-particulate pellet formulation.

AIM: Quantitative evaluation of the effect caused by vaginal administration of gelatin capsules loaded with starch pellets and lyophilized powder, respectively, on vaginal pH and microflora. METHOD: Administration of gelatin capsules loaded with fast-disintegrating starch pellets (group P) or lyophilized lactose/skimmed milk (group L) was compared to no intervention (group C) in a 3-way randomized, double-blinded, parallel study with 18 volunteers. Follow-up visits were at day 6 (immediately after administration), day 14 (pill stop), day 22 (after withdrawal bleeding) and day 35 (midcycle). Vaginal pH was measured and swabs were taken for Gram staining and culture to assess the presence of hydrogen peroxide-producing lactobacilli. Colposcopy was performed to assess the occurrence of adverse effects on the vaginal and ectocervical mucosa. RESULTS: No severe adverse events occurred. For all women, vaginal pH and Gram stain were normal from screening until pill stop. Although immediately after withdrawal bleeding, 8 out of 18 women had an elevated pH, a disturbed microflora or lacked hydrogen peroxide-producing lactobacilli, all women had hydrogen peroxide-producing lactobacilli and a normal vaginal pH at midcycle, and all but two had a normal Gram stain. CONCLUSION: No major differences could be observed between the groups, whereby all changes in pH and microflora could be ascribed to withdrawal bleeding, indicating that gelatin capsules, starch pellets and lyophilized powder are acceptable carrier materials for the vaginal delivery of probiotic strains.