The profiling of microbiota in vaginal swab samples using 16S rRNA gene sequencing and IS-pro analysis.

BACKGROUND: 16S rRNA gene sequencing is currently the most common way of determining the composition of microbiota. This technique has enabled many new discoveries to be made regarding the relevance of microbiota to the health and disease of the host. However, compared to other diagnostic techniques, 16S rRNA gene sequencing is fairly costly and labor intensive, leaving room for other techniques to improve on these aspects. RESULTS: The current study aimed to compare the output of 16S rRNA gene sequencing to the output of the quick IS-pro analysis, using vaginal swab samples from 297 women of reproductive age. 16S rRNA gene sequencing and IS-pro analyses yielded very similar vaginal microbiome profiles, with a median Pearson's R2 of 0.97, indicating a high level of similarity between both techniques. CONCLUSIONS: We conclude that the results of 16S rRNA gene sequencing and IS-pro are highly comparable and that both can be used to accurately determine the vaginal microbiota composition, with the IS-pro analysis having the benefit of rapidity.

The Y-chromosome F haplogroup contributes to the development of Barrett's esophagus-associated esophageal adenocarcinoma in a white male population.

Barrett's esophagus (BE) is a metaplastic condition of the distal esophagus, resulting from longstanding gastroesophageal reflux disease (GERD). BE predisposes for the highly malignant esophageal adenocarcinoma (EAC). Both BE and EAC have the highest frequencies in white males. Only a subset of patients with GERD develop BE, while <0.5% of BE will progress to EAC. Therefore, it is most likely that the development of BE and EAC is associated with underlying genetic factors. We hypothesized that in white males, Y-chromosomal haplogroups are associated with BE and EAC. To investigate this we conducted a multicenter study studying the frequencies of the Y-chromosomal haplogroups in GERD, BE, and EAC patients. We used genomic analysis by polymerase chain reaction and restriction fragment length polymorphism to determine the frequency of six Y-chromosomal haplogroups (DE, F(xJ,xK), K(xP), J, P(xR1a), and R1a) between GERD, BE, and EAC in a cohort of 1,365 white males, including 612 GERD, 753 BE patients, while 178 of the BE patients also had BE-associated EAC. Univariate logistic regression analysis was used to compare the outcomes. In this study, we found the R1a (6% vs. 9%, P = 0.04) and K (3% vs. 6%, P = 0.035) to be significantly underrepresented in BE patients as compared to GERD patients with an odds ratio (OR) of 0.63 (95% CI 0.42-0.95, P = 0.03) and of 0.56 (95% CI 0.33-0.96, P = 0.03), respectively, while the K haplogroup was protective against EAC (OR 0.30; 95% CI 0.07-0.86, P = 0.05). A significant overrepresentation of the F haplogroup was found in EAC compared to BE and GERD patients (34% vs. 27% and 23%, respectively). The F haplogroup was found to be a risk factor for EAC with an OR of 1.5 (95% CI 1.03-2.19, P = 0.03). We identified the R1a and K haplogroups as protective factors against development of BE. These haplogroups have low frequencies in white male populations. Of importance is that we could link the presence of the predominantly occurring F haplogroup in white males to EAC. It is possible that this F haplogroup is associated to genetic variants that predispose for the EAC development. In future, the haplogroups could be applied to improve stratification of BE and GERD patients with increased risk to develop BE and/or EAC.

The vaginal microbiome as a predictor for outcome of in vitro fertilization with or without intracytoplasmic sperm injection: a prospective study.

STUDY QUESTION: Is the presence or absence of certain vaginal bacteria associated with failure or success to become pregnant after an in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (IVF-ICSI) treatment? SUMMARY ANSWER: Microbiome profiling with the use of interspace profiling (IS-pro) technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. WHAT IS KNOWN ALREADY: Live-birth rates for an IVF or IVF-ICSI treatment vary between 25 and 35% per cycle and it is difficult to predict who will or will not get pregnant after embryo transfer (ET). Recently, it was suggested that the composition of the vaginal microbiota prior to treatment might predict pregnancy outcome. Analysis of the vaginal microbiome prior to treatment might, therefore, offer an opportunity to improve the success rate of IVF or IVF-ICSI. STUDY DESIGN, SIZE, DURATION: In a prospective cohort study, 303 women (age, 20-42 years) undergoing IVF or IVF-ICSI treatment in the Netherlands were included between June 2015 and March 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Study subjects provided a vaginal sample before the start of the IVF or IVF-ICSI procedure. The vaginal microbiota composition was determined using the IS-pro technique. IS-pro is a eubacterial technique based on the detection and categorization of the length of the 16S-23S rRNA gene interspace region. Microbiome profiles were assigned to community state types based on the dominant bacterial species. The predictive accuracy of the microbiome profiles for IVF and IVF-ICSI outcome of fresh ET was evaluated by a combined prediction model based on a small number of bacterial species. From this cohort, a model was built to predict outcome of fertility treatment. This model was externally validated in a cohort of 50 women who were undergoing IVF or IVF-ICSI treatment between March 2018 and May 2018 in the Dutch division of the MVZ VivaNeo Kinderwunschzentrum Düsseldorf, Germany. MAIN RESULTS AND THE ROLE OF CHANCE: In total, the vaginal microbiota of 192 women who underwent a fresh ET could be analysed. Women with a low percentage of Lactobacillus in their vaginal sample were less likely to have a successful embryo implantation. The prediction model identified a subgroup of women (17.7%, n = 34) who had a low chance to become pregnant following fresh ET. This failure was correctly predicted in 32 out of 34 women based on the vaginal microbiota composition, resulting in a predictive accuracy of 94% (sensitivity, 26%; specificity, 97%). Additionally, the degree of dominance of Lactobacillus crispatus was an important factor in predicting pregnancy. Women who had a favourable profile as well as <60% L. crispatus had a high chance of pregnancy: more than half of these women (50 out of 95) became pregnant. In the external validation cohort, none of the women who had a negative prediction (low chance of pregnancy) became pregnant. LIMITATIONS, REASONS FOR CAUTION: Because our study uses a well-defined study population, the results will be limited to the IVF or IVF-ICSI population. Whether these results can be extrapolated to the general population trying to achieve pregnancy without ART cannot be determined from these data. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that vaginal microbiome profiling using the IS-pro technique enables stratification of the chance of becoming pregnant prior to the start of an IVF or IVF-ICSI treatment. Knowledge of their vaginal microbiota may enable couples to make a more balanced decision regarding timing and continuation of their IVF or IVF-ICSI treatment cycles. STUDY FUNDING/COMPETING INTEREST(S): This study was financed by NGI Pre-Seed 2014-2016, RedMedTech Discovery Fund 2014-2017, STW Valorisation grant 1 2014-2015, STW Take-off early phase trajectory 2015-2016 and Eurostars VALBIOME grant (reference number: 8884). The employer of W.J.S.S.C. has in collaboration with ARTPred acquired a MIND subsidy to cover part of the costs of this collaboration project. The following grants are received but not used to finance this study: grants from Innovatie Prestatie Contract, MIT Haalbaarheid, other from Dutch R&D tax credit WBSO, RedMedTech Discovery Fund, (J.D.d.J.). Grants from Ferring (J.S.E.L., K.F., C.B.L. and J.M.J.S.S.), Merck Serono (K.F. and C.B.L.), Dutch Heart Foundation (J.S.E.L.), Metagenics Inc. (J.S.E.L.), GoodLife (K.F.), Guerbet (C.B.L.). R.K. is employed by ARTPred B.V. during her PhD at Erasmus Medical Centre (MC). S.A.M. has a 100% University appointment. I.S.P.H.M.S., S.A.M. and A.E.B. are co-owners of IS-Diagnostics Ltd. J.D.d.J. is co-owner of ARTPred B.V., from which he reports personal fees. P.H.M.S. reports non-financial support from ARTPred B.V. P.H.M.S., J.D.d.J. and A.E.B. have obtained patents `Microbial population analysis' (9506109) and `Microbial population analysis' (20170159108), both licenced to ARTPred B.V. J.D.d.J. and A.E.B. report patent applications `Method and kit for predicting the outcome of an assisted reproductive technology procedure' (392EPP0) and patent `Method and kit for altering the outcome of an assisted reproductive technology procedure' by ARTPred. W.J.S.S.C. received personal consultancy and educational fees from Goodlife Fertility B.V. J.S.E.L. reports personal consultancy fees from ARTPred B.V., Titus Health B.V., Danone, Euroscreen and Roche during the conduct of the study. J.S.E.L. and N.G.M.B. are co-applicants on an Erasmus MC patent (New method and kit for prediction success of in vitro fertilization) licenced to ARTPred B.V. F.J.M.B. reports personal fees from Advisory Board Ferring, Advisory Board Merck Serono, Advisory Board Gedeon Richter and personal fees from Educational activities for Ferring, outside the submitted work. K.F. reports personal fees from Ferring (commercial sponsor) and personal fees from GoodLife (commercial sponsor). C.B.L. received speakers' fee from Ferring. J.M.J.S.S. reports personal fees and other from Merck Serono and personal fees from Ferring, unrelated to the submitted paper. The other authors declare that they have no competing interests. TRIAL REGISTRATION NUMBER: ISRCTN83157250. Registered 17 August 2018. Retrospectively registered.

Study protocol: The Dutch 20|30 Postmeningitis study: a cross-sectional follow-up of two historical childhood bacterial meningitis cohorts on long-term outcomes.

BACKGROUND: Bacterial meningitis (BM) is a serious, life-threatening infectious disease of the central nervous system that often occurs in young children. The most common severe to moderate sequelae following BM are sensorineural hearing loss, neuromotor disabilities and mental retardation, while subtle sequelae include academic and behavioral disabilities. It is largely unknown whether these more subtle sequelae persist into adolescence and adulthood. Therefore, this study will investigate the very long-term effects of childhood BM in later life. Better understanding of long-term effects and early identification of adverse outcomes after BM are essential for more timely interventions. Additionally, certain single nucleotide polymorphisms (SNPs) are associated with disease severity and might predict adverse sequelae. These include SNPs in genes encoding for pathogen recognition and immune response upon infection. Accordingly, a secondary objective of this study is to investigate the role of genetic variation in BM and use any insights to predict short- and long-term outcomes. METHODS: In the Dutch 20|30 Postmeningitis study, adolescents and young adults (n = 947) from two historical cohorts with a prior episode of BM during childhood will be enrolled into a cross-sectional follow-up investigation using mainly questionnaires that examine executive and behavioral functioning, health-related quality of life, subjective hearing, mood and sleeping disorders, academic performance, and economic self-sufficiency. The results will be compared to normative data by one-sample t-tests. Multivariable regression analysis will be used to assess for any associations with causative pathogens and severity of BM. Participants that complete the questionnaires will be approached to provide a swab for buccal DNA and subsequent sequencing analyses. Logistic regression models will be used to predict sequelae. DISCUSSION: The unique follow-up duration of this cohort will enable us to gain insights into the possible very long-term adverse effects of childhood BM and how these might impact on quality of life. The investigation of host genetic factors will contribute to the development of prediction models which will serve as prognostic tools to identify children who are at high risk of adverse outcome after BM. TRIAL REGISTRATION: Dutch Trial Register NTR-6891. Retrospectively registered 28 December 2017.

The Netherlands Chlamydia cohort study (NECCST) protocol to assess the risk of late complications following Chlamydia trachomatis infection in women.

BACKGROUND: Chlamydia trachomatis (CT), the most common bacterial sexually transmitted infection (STI) among young women, can result in serious sequelae. Although the course of infection is often asymptomatic, CT may cause pelvic inflammatory disease (PID), leading to severe complications, such as prolonged time to pregnancy, ectopic pregnancy, and tubal factor subfertility. The risk of and risk factors for complications following CT-infection have not been assessed in a long-term prospective cohort study, the preferred design to define infections and complications adequately. METHODS: In the Netherlands Chlamydia Cohort Study (NECCST), a cohort of women of reproductive age with and without a history of CT-infection is followed over a minimum of ten years to investigate (CT-related) reproductive tract complications. This study is a follow-up of the Chlamydia Screening Implementation (CSI) study, executed between 2008 and 2011 in the Netherlands. For NECCST, female CSI participants who consented to be approached for follow-up studies (n = 14,685) are invited, and prospectively followed until 2022. Four data collection moments are foreseen every two consecutive years. Questionnaire data and blood samples for CT-Immunoglobulin G (IgG) measurement are obtained as well as host DNA to determine specific genetic biomarkers related to susceptibility and severity of infection. CT-history will be based on CSI test outcomes, self-reported infections and CT-IgG presence. Information on (time to) pregnancies and the potential long-term complications (i.e. PID, ectopic pregnancy and (tubal factor) subfertility), will be acquired by questionnaires. Reported subfertility will be verified in medical registers. Occurrence of these late complications and prolonged time to pregnancy, as a proxy for reduced fertility due to a previous CT-infection, or other risk factors, will be investigated using longitudinal statistical procedures. DISCUSSION: In the proposed study, the occurrence of late complications following CT-infection and its risk factors will be assessed. Ultimately, provided reliable risk factors and/or markers can be identified for such late complications. This will contribute to the development of a prognostic tool to estimate the risk of CT-related complications at an early time point, enabling targeted prevention and care towards women at risk for late complications. TRIAL REGISTRATION: Dutch Trial Register NTR-5597 . Retrospectively registered 14 February 2016.

Vaginal high-risk human papillomavirus infection in a cross-sectional study among women of six different ethnicities in Amsterdam, the Netherlands: the HELIUS study.

OBJECTIVE: In the Netherlands the incidence of cervical cancer is higher among ethnic minority populations compared with the general Dutch population. We investigated the prevalence of, and risk factors associated with, vaginal high-risk human papillomavirus (hrHPV) infection in women of six different ethnicities living in Amsterdam. METHODS: For this cross-sectional study we selected women aged 18-34 years old of six ethnicities from the large-scale multiethnic HEalthy LIfe in an Urban Setting study. Self-collected vaginal swabs were tested for HPV DNA and genotyped using a highly sensitive PCR and reverse line blot assay (short PCR fragment (SPF)10-PCR DNA enzyme immunoassay/LiPA25-system version-1, delft diagnostic laboratory (DDL)). Participants completed a questionnaire regarding demographics and sexual behaviour. Logistic regression using generalised estimating equations was used to assess risk factors of hrHPV, and to investigate whether prevalence of hrHPV differed among ethnicities. RESULTS: The study population consisted of 592 women with a median age of 27 (IQR: 23-31) years. Dutch and African Surinamese women reported the highest sexual risk behaviour. HrHPV prevalence was highest in the Dutch (40%) followed by the African Surinamese (32%), Turkish (29%), Ghanaian (26%), Moroccan (26%) and South-Asian Surinamese (18%). When correcting for sexual risk behaviour, the odds to be hrHPV-positive were similar for all non-Dutch groups when compared with that of the Dutch group. CONCLUSIONS: We found an overall higher hrHPV prevalence and higher sexual risk behaviour in the native Dutch population. Further research is needed to unravel the complex problem concerning cervical cancer disparities, such as differences in participation in the cervical cancer screening programme, or differences in clearance and persistence of hrHPV.

Chlamydia trachomatis IgG seroprevalence in the general population of the Netherlands in 1996 and in 2007: differential changes by gender and age.

OBJECTIVES: Chlamydia trachomatis (CT) reporting rates from sexually transmitted infection clinics and general practitioners have shown a rising trend in the Netherlands. It is unknown to what extent this reflects increased CT transmission or improved case finding. To achieve more insight into the CT epidemic, we explored the CT IgG seroprevalence (a marker of past CT infection) in the general population of the Netherlands in 1996 and in 2007. METHODS: From two population-based studies in 1996 and 2007, serum samples, demographic and sexual behaviour outcomes were examined, including 1246 men and 1930 women aged 15-39 years. Serum CT IgG antibodies were analysed using the Medac CT IgG ELISA test. Multivariate logistic regression analyses explored the seroprevalence and determinants over time. RESULTS: The CT IgG seroprevalence was higher in women than in men (10% vs 6%). Among women aged 25-39 years the seroprevalence was lower in 2007 (9%) than in 1996 (14%; adjusted OR (aOR) 0.6, 95% CI 0.4 to 0.8). There was no statistical evidence of a difference in seroprevalence within birth cohorts. Factors associated with seropositivity were male gender (aOR 0.4, 95% CI 0.3 to 0.7), a self-reported history of CT infection (aOR 5.1, 95% CI 2.6 to 10.0), age 25-39 years (aOR 1.7, 95% CI 1.1 to 2.7), non-Western ethnicity (aOR 2.2, 95% CI 1.4 to 3.3) and ≥ 2 recent sexual partners (aOR 2.2, 95% CI 1.3 to 3.5). CONCLUSIONS: Between 1996 and 2007 the proportion of individuals in the general population with CT IgG antibodies was lower among women aged 25-39 years, but remained similar among younger women and men.

Serogroup distribution of urogenital Chlamydia trachomatis in urban ethnic groups in The Netherlands.

The prevalence of Chlamydia trachomatis varies between ethnic groups in The Netherlands. It is, however, unknown whether this is associated with specific serogroups. The objective of this study was to determine whether serogroup distribution is associated with ethnic origin in the region of The Hague, The Netherlands. Serogroups of 370 microbiologically confirmed C. trachomatis-positive samples were analysed. The samples were obtained from 247 women and 123 men between January and October 2008, of self-reported Dutch Caucasian, Dutch Antillean, Surinamese, N. African/Turkish or other descent. We observed a difference in serogroup distribution comparing Dutch Caucasian women to Dutch Antillean women (χ2 for distribution P = 0·035). Serogroup C was more common in Dutch Antillean women, whereas serogroup B was less common (P = 0·03). This difference was not observed for Dutch Antillean men. The observed difference in distribution of C. trachomatis serogroups between ethnic groups is relevant for further transmission studies.

Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis.

Reports of potential treatment failure have raised particular concerns regarding the efficacy of the single dose azithromycin regimen in the treatment of urogenital and anorectal Chlamydia trachomatis (CT) infections. Several factors have been suggested, including heterotypic resistance. Antimicrobial susceptibility testing in CT requires cell culture with serial dilutions of antibiotics, which is laborious and for which there is no standardized testing methodology. One method to partly overcome these difficulties would be to use a genotypic resistance assay, however most current available assays do still require prior CT culture. In order to facilitate the assessment of genotypic resistance directly from clinical samples, without the need for prior culture, the aim of this study was to develop a CT specific PCR assay for the assessment of resistance associated mutations (RAMs) in the 23S rRNA gene, and to evaluate a sample of clinical cases in which CT PCR's remained positive during follow-up despite azithromycin treatment. Neither the in silico analysis nor the analytical specificity testing demonstrated clinically relevant cross-reactivity with other bacterial species. These results in conjunction with the analytical sensitivity demonstrating consistent CT 23S rRNA gene detection in the range of 10e3 IFU/mL, exemplify the assay's apt performance. Although no known macrolide RAMs were detected in the clinical cases, the described assay allows future culture independent macrolide RAM surveillance in CT, and increases accessibility for other laboratories to engage in screening.

Genetic variation of innate immune response genes in invasive pneumococcal and meningococcal disease applied to the pathogenesis of meningitis.

The susceptibility, severity and prognosis of infectious diseases depend on the ability of the host immune system to respond to pathogens. Genetic variation of immune response genes is associated with susceptibility to and severity of infectious diseases. Bacterial meningitis (BM) is a serious and life-threatening infectious disease of the central nervous system (CNS). Despite adequate antibiotic treatment and immunization strategies, mortality remains high, especially in developing countries. Streptococcus pneumoniae and Neisseria meningitidis are the two most common causative microorganisms of BM worldwide. The pathogenesis of BM starts with mucosal bacterial colonization, followed by invasion and survival of bacteria in the bloodstream, crossing of the blood-brain barrier, finally causing infection in the CNS, where host defense is less adequate. Host defense to BM starts with a complex cascade of pathogen recognition and subsequent intracellular signaling causing transcription of genes leading to the production of inflammatory mediators. Although this immune reaction is essential for killing microbes, it is also associated with damage to healthy cells and thus adverse disease outcome. This review provides an overview of the pathogenesis of invasive pneumococcal disease and invasive meningococcal disease related to the influence of genetic variation in genes involved in innate immunity, focusing on BM.

Analyses of multiple-site and concurrent Chlamydia trachomatis serovar infections, and serovar tissue tropism for urogenital versus rectal specimens in male and female patients.

OBJECTIVES: The aims of this study were: to determine the incidence of concurrent infections on a serovar level; to determine the incidence of multiple anatomical infected sites on a detection and genotyping level and analyse site-specific serovar distribution; to identify tissue tropism in urogenital versus rectal specimens. METHODS: Chlamydia trachomatis-infected patients in two populations were analysed: 75 visiting the outpatient department of obstetrics and gynaecology of the MC Haaglanden, and 358 visiting the outpatient sexually transmitted disease clinic, The Hague, The Netherlands. The PACE 2 assay (Gen-Probe) was used to detect C trachomatis from urethral, cervical, vaginal, oropharyngeal and anorectal swabs. C trachomatis genotyping was performed on all C trachomatis positive samples, using the CT-DT genotyping assay. RESULTS: Samples from 433 patients (256 female and 177 male) with confirmed C trachomatis infection were analysed. In 11 patients (2.6%), concurrent serovars in one anatomical sample site were present. In 62 (34.1%) female and four (9.3%) male patients, multiple sample site infections were found. A substantial percentage of women tested at the cervical/vaginal and rectal site were found to be positive at both sites (36.1%, 22/61). In men, D/Da and G/Ga serovars were more prevalent in rectal than urogenital specimens (p=0.0081 and p=0.0033, respectively), while serovar E was more prevalent in urogenital specimens (p=0.0012). CONCLUSIONS: The prevalence of multiple serovar infections is relatively low. Significant differences in serovar distribution are found in rectal specimens from men, with serovar G/Ga being the most prominent, suggesting tissue tropism.

Comparison of three genotyping methods to identify Chlamydia trachomatis genotypes in positive men and women.

Chlamydia trachomatis (Ct) comprises 3 serogroups and 19 serovars. Different genotyping methods are available to differentiate between the serovars. The aim of this study was to evaluate the sensitivity and discriminatory power of three genotyping methods, respectively Omp1 sequencing, the Ct Detection and genoTyping (DT) assay and the pmpH real-time PCR discriminating an LGV infection from a non-LGV infection. In total, 50 Aptima Combo 2 (AC2) Ct positive samples were selected and tested with the 3 genotyping methods. The Ct-DT assay detected 3 double Ct infections that caused a non interpretable result by Omp1 sequencing, while Omp1 sequencing has a higher discriminatory power that gave additional information about Ct genovariants. All three methods detected the 6 LGV samples. Although the pmpH real-time PCR detected all LGV infections, a substantial amount (24%) of non-LGV infections were missed. The sensitivity compared to AC2 Ct detection was 80% (95% CI 67-89%) for the Ct-DT assay, 72% (95% CI 58-83%) for Omp1 sequencing and 64% (95% CI 50-76%) for the pmpH real-time PCR. In conclusion, the Ct-DT assay is appropriate for serovar distribution studies, epidemiological studies and differentiation between an LGV and non-LGV Ct infection, while Omp1 sequencing is more appropriate for phylogenetic studies. The pmpH real-time PCR is suitable as second assay to differentiate between an LGV and non-LGV infection, but not as primary detection assay, due to its low sensitivity for non-LGV strains.

NOD2, CD14 and TLR4 mutations do not influence response to adalimumab in patients with Crohn's disease: a preliminary report.

INTRODUCTION: Adalimumab is a recombinant fully-human monoclonal immunoglobulin (IgG1) antibody utilized in the treatment of Crohn's disease. Unfortunately no clinical or genetic markers exist to predict response to anti-tumor necrosis factor-alpha (TNF) therapy. The aim of this study was to evaluate the association between selected genes involved in cytokine regulation and response to adalimumab treatment in Crohn's disease. METHODS: twenty-four patients with Crohn's disease either naïve (n = 8) or had lost response or were unable to tolerate the chimeric anti-TNF antibody infliximab (n=16) were enrolled in the study. Patients were genotyped for main polymorphisms in NOD2, CD14 and TLR4 genes. Response to adalimumab treatment was defined as a decrease of Crohn's disease activity index of at least 100 points or a closure of at least 50% of fistulas in case of fistulizing Crohn's disease. RESULTS: overall, 75% of patients did respond to treatment. However, no statistically significant association was found between any of the genotypes and the response to adalimumab. CONCLUSIONS: In our small study group no association between the studied polymorphisms and response to adalimumab was apparent. Systematic studies to search for genetic markers of response to anti-TNF therapy are necessary.

Limited presence of Waddlia chondrophila in drinking water systems in the Netherlands.

Waddlia chondrophila is an emerging pathogen belonging to the order of Chlamydiales. This obligate intracellular bacterium was initially isolated from an aborted bovine fetus and is associated with adverse pregnancy outcomes in women. The ability of W. chondrophila to reside and replicate within a range of free-living amoebae implies a possible widespread environmental presence. Potential hosts of W. chondrophila are present in Dutch drinking water. This study therefore investigated the presence of W. chondrophila DNA in drinking water by analysing 59 samples from ten drinking water systems throughout the Netherlands. Samples were taken at three distances from the treatment plant, during both summer and winter. Twelve of the samples were positive, originating from two of the treatment plants, of which three samples were quantifiable.

The relation of the vaginal microbiota to early pregnancy development during in vitro fertilization treatment-A meta-analysis.

An abnormal vaginal microbiota composition has been shown to lead to pre-term births, miscarriage, and problems with conceiving. Studies have suggested that dysbiosis reduces successful early pregnancy development during IVF. However, conflicting reports exist. This meta-analysis aims to answer the following question: what is the aggregated effect found by studies investigating the influence of the vaginal microbiota composition on early pregnancy rates after IVF treatment? A systematic review was performed using the Medline and EMBASE databases, using search terms for healthy vaginal microbiota, abnormal vaginal microbiota, fertility and pregnancy. The search resulted in six included articles. Of these, all six were used for further meta-analysis. The main outcome measures were the clinical pregnancy rate, determined through ultrasound proven fetal heartbeat and/or hCG results before 10 weeks gestation, in relation to the vaginal microbiota composition. We found a correlation between abnormal vaginal microbiota and lower rates of early pregnancy development after IVF treatment (OR = 0.70, 95% CI = 0.49 - 0.99). One study showed the reverse correlation. However, heterogeneity between study methodologies in various forms was found. In conclusion, women with an abnormal vaginal microbiota are roughly 1.4 times less likely to have a successful early pregnancy development after IVF treatment when compared to women with normal microbiota.

Increased infiltration of Chlamydophila pneumoniae in the vessel wall of human veins after perfusion.

BACKGROUND: Several studies have suggested an association between Chlamydophila pneumoniae (Cp) infection and atherosclerosis. A recent study detected Cp DNA in the saphenous vein of 12% of all patients before bypass grafting and in 38% of failed grafts. We used a system in which human veins were perfused with autologous blood under arterial pressure. MATERIALS AND METHODS: Veins were surplus segments of saphenous veins of coronary artery bypass grafting (CABG) patients. Vein grafts were perfused with the blood of the same patient after CABG procedures. Veins were analysed for Cp-specific membrane protein using immunohistochemical and PCR analysis. Veins were analysed before and after perfusion (up to 4 h). The number of Cp positive cells was then quantified in the vein layers. RESULTS: Cp protein was detected within macrophages only. In non-perfused veins, Cp was present in the adventitia in 91% of all patients, in the circular (64%) and longitudinal (23%) layer of the media. No positivity was found in the intima. Perfusion subsequently resulted in a significant increase of Cp positive cells within the circular layer of the media that, however, differed strongly between different patients. Cp DNA was not detected by PCR in those specimens. CONCLUSION: Cp protein was present in 91% of veins, but the number of positive cells differed remarkably between patients. Perfusion of veins resulted in increased infiltration of Cp into the circular layer. These results may point to a putative discriminating role of Cp with respect to graft failure between different patients.

Host and microbiological factors related to dental caries development.

Studies on dental caries suggest that in severe cases it may induce a systemic immune response. This occurs particularly when caries progresses into pulpal inflammation and results in abscess or fistula formation (AFF). We hypothesized that severe dental caries will affect the general health of children. The acute phase proteins alpha-1-acid glycoprotein (AGP), C-reactive protein (CRP) and the cytokine neopterin were chosen as parameters to monitor general health. Also, a polymorphism in the bacterial ligand CD14 (-260) was studied to investigate the relationship between genotype sensitivity for bacterial infections and AFF. In Suriname, children aged 6 years were recruited and enrolled into a dental care scheme, randomly assigned to 4 groups with different treatment strategies and monitored longitudinally. 348 children were included in the present study. Blood and saliva samples were taken at baseline and 1 year, and concentrations of serum AGP, CRP, neopterin, salivary Streptococcus mutans and CD14-260 C>T polymorphism were determined. There was no significant association between different treatment strategies and the serum parameters. Binary logistic regression analyses revealed a significant association between AFF as the outcome variable and the CD14 genotype and the concentrations of CRP and of neopterin as factors (p < 0.05). A significant negative association was found between the CD14-260 TT and AFF (p = 0.035, OR = 3.3) for the whole population. For children who had 4 or more carious lesions at baseline, the significance increased (p = 0.005, OR = 4.8), suggesting that the CD14-260 TT genotype was protective for AFF as a consequence of dental caries.

Comparison of the Mikrogen multi-target ELISA with the Mikrogen recomLine immunoblot for the detection of Chlamydia trachomatis IgG antibodies in serum in infertile women.

OBJECTIVES: Chlamydia trachomatis (CT) IgG serology is used in many fertility clinics in order to estimate the risk for tubal factor infertility (TFI) in the fertility work-up. The predictive value for TFI of the currently used mono-target CT serology test should be improved. This study compares the performance of the new multi-target Mikrogen recomWell CT IgG ELISA with the Mikrogen recomLine CT immunoblot and visualizes distribution of individual antibodies in serum with the immunoblot in order to potentially improve the current CT IgG serology test that is clinically used. METHODS: Study population consisted of 183 Dutch Caucasian infertile women who underwent laparoscopy and/or hysterosalpingography. 48 women had TFI, 135 were controls. Serum was tested with Mikrogen CT IgG ELISA, which detects 3 CT IgG antibodies in one well, and Mikrogen CT immunoblot, which can individually detect 5 CT IgG antibodies. Tests were compared based on the results in general and in the case and control group also taking the individual antibodies into account. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), Kappa value and distribution of individual antibodies in positive samples were calculated. RESULTS: In 183 patients 51% tested positive in the ELISA versus 35% in the immunoblot. 32% versus 65% tested negative. Difference between PPV was not statistically significant (33% and 39% respectively) and NPV in both tests was 81%. Difference in sensitivity and specificity was statistically significant, respectively 65% vs. 52% and 54% vs. 71%. Kappa was only 45%. 64.5% of samples that tested positive with ELISA were positive for at least 4 individual CT antibodies with the immunoblot. CONCLUSION: The concordance between CT ELISA and CT immunoblot is moderate. Due to separate criteria for positivity of both tests there is a significant difference in sensitivity and specificity. PPV and NPV, the most relevant characteristics for clinicians, of both tests did not differ significantly. The distribution of individual antibodies and the adjustment of the immunoblot algorithm will be further explored in the future in order to develop a potentially better prediction method for TFI with a higher clinical accuracy.

Chlamydia trachomatis antibody detection in home-collected blood samples for use in epidemiological studies.

Capillary blood collected in serum tubes was subjected to centrifugation delay while stored at room temperature. Chlamydia trachomatis (CT) IgG concentrations in aliquoted serum of these blood samples remained stable for seven days after collection. CT IgG concentrations can reliably be measured in mailed blood samples in epidemiological studies.