Bacterial communities in penile skin, male urethra, and vaginas of heterosexual couples with and without bacterial vaginosis

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Microbiome 4 (2016): 16, doi:10.1186/s40168-016-0161-6.The epidemiology of bacterial vaginosis (BV) suggests it is sexually transmissible, yet no transmissible agent has been identified. It is probable that BV-associated bacterial communities are transferred from male to female partners during intercourse; however, the microbiota of sexual partners has not been well-studied. Pyrosequencing analysis of PCR-amplified 16S rDNA was used to examine BV-associated bacteria in monogamous couples with and without BV using vaginal, male urethral, and penile skin specimens. The penile skin and urethral microbiota of male partners of women with BV was significantly more similar to the vaginal microbiota of their female partner compared to the vaginal microbiota of non-partner women with BV. This was not the case for male partners of women with normal vaginal microbiota. Specific BV-associated species were concordant in women with BV and their male partners. In monogamous heterosexual couples in which the woman has BV, the significantly higher similarity between the vaginal microbiota and the penile skin and urethral microbiota of the male partner, supports the hypothesis that sexual exchange of BV-associated bacterial taxa is common.This work was supported by National Institute of Health Grant R01 AI079071-01A1

Social Perceptions of Forest Ecosystem Services in the Democratic Republic of Congo

The forests of the Albertine Rift are known for their high biodiversity and the important ecosystem services they provide to millions of inhabitants. However, their conservation and the maintenance of ecosystem service delivery is a challenge, particularly in the Democratic Republic of the Congo. Our research investigates how livelihood strategy and ethnicity affects local perceptions of forest ecosystem services. We collected data through 25 focus-group discussions in villages from distinct ethnic groups, including farmers (Tembo, Shi, and Nyindu) and hunter-gatherers (Twa). Twa identify more food-provisioning services and rank bush meat and honey as the most important. They also show stronger place attachment to the forest than the farmers, who value other ecosystem services, but all rank microclimate regulation as the most important. Our findings help assess ecosystem services trade-offs, highlight the important impacts of restricted access to forests resources for Twa, and point to the need for developing alternative livelihood strategies for these communities

Evaluation of presumably disease causing SCN1A variants in a cohort of common epilepsy syndromes

Objective: The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods: We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation: We identified 8 known missense mutations, previously reported as path

A novel whole-bacterial enzyme linked-immunosorbant assay to quantify Chlamydia trachomatis specific antibodies reveals distinct differences between systemic and genital compartments.

Chlamydia trachomatis (CT) is the leading sexually transmitted bacterial infection. The continued global burden of CT infection strongly predicates the need for a vaccine to supplement current chlamydial control programs. The correlates of protection against CT are currently unknown, but they must be carefully defined to guide vaccine design. The localized nature of chlamydial infection in columnar epithelial cells of the genital tract necessitates investigation of immunity at the site of infection. The purpose of this study was to develop a sensitive whole bacterial enzyme-linked immunosorbent assay (ELISA) to quantify and compare CT-specific IgG and IgA in sera and genital secretions from CT-infected women. To achieve this, elementary bodies (EBs) from two of the most common genital serovars (D and E) were attached to poly-L-lysine-coated microtiter plates with glutaraldehyde. EB attachment and integrity were verified by the presence of outer membrane antigens and the absence of bacterial cytoplasmic antigens. EB-specific IgG and IgA standards were developed by pooling sera with high titers of CT-specific antibodies from infected women. Serum, endocervical and vaginal secretions, and endocervical cytobrush specimens from CT-infected women were used to quantify CT-specific IgG and IgA which were then normalized to total IgG and IgA, respectively. Analyses of paired serum and genital samples revealed significantly higher proportions of EB-specific antibodies in genital secretions compared to sera. Cervical and vaginal secretions and cytobrush specimens had similar proportions of EB-specific antibodies, suggesting any one of these genital sampling techniques could be used to quantify CT-specific antibodies when appropriate normalization methodologies are implemented. Overall, these results illustrate the need to investigate genital tract CT antibody responses, and our assay provides a useful quantitative tool to assess natural immunity in defined clinical groups and CT vaccine trials

Bacterial communities in heterosexual couples

<p>Bacterial tag-encoded FLX amplicon pyrosequencing analysis of the V4 -V6 region of the 16S rRNA gene using primers 530F: GTGCCAGCMGCNGCGG and 1100R: GGGTTNCGNTCGTTG. Sequence files are in FASTA format.</p> <p>DNA was extracted from penile skin (PS), urethral (UT) and vaginal (VG) specimens from normal (N) women and women with bacterial vaginosis (BV) and their sexual partners.</p

EB IgG and IgA specific activity in serum and genital tract secretions in CT-infected women.

<p><b>(A)</b> EB IgG and <b>(B)</b> IgA specific activity in matched serum, endocervical and vaginal secretions, and cytobrush samples were determined by dividing the anti-EB IgG or IgA antibody (in ng/ml) by the respective total IgG or IgA concentration (in μg/ml) in each sample. Dashed lines denote the thresholds for significance (mean specific activity + 3 SD of negative controls). *p < 0.05 and **p < 0.01 by two-tailed Wilcoxon matched pairs rank sum test.</p

Creation of EB IgG and IgA standards for antibody quantitation.

<p>Sera from 20 CT-infected women were screened at a 1/1,000 dilution for <b>(A)</b> IgG antibodies and at a 1/50 dilution for <b>(B)</b> IgA antibodies that bound to pooled EBs. Shown is the mean absorbance ± SD for duplicate samples. The colored columns indicate sera that were subsequently pooled for use as the EB IgG or IgA standard and calibrated relative to total IgG and IgA standards, as described in the Methods. The dashed line represents the mean absorbance + 3 SD obtained with negative control IgG proteins at 10–20 μg/ml or IgA proteins at 20–40 μg/ml, concentrations approximately equivalent to those that would be present in serum diluted 1/1,000 (for IgG) or 1/50 (for IgA). <b>(C)</b> The EB IgG standard curve obtained after pooling the indicated sera is shown as mean ± SD for duplicate 2-fold serial dilutions, starting at a 1/1,000 dilution and a concentration of 19 ng/ml. <b>(D)</b> The EB IgA standard curve is similarly shown as mean ± SD for duplicate 2-fold dilutions starting at a 1/50 dilution and a concentration of 9.6 ng/ml. Using the EB IgG and IgA standards, <b>(E)</b> anti-EB IgG and <b>(F)</b> anti-EB IgA antibodies were measured in serum from 10 CT-infected women. Shown is the mean concentration ± SD obtained in two separate experiments. The inter-assay deviation for each sample is shown as the % CV (100% x SD/mean) above each column.</p

Chlamydial antigens measured on captured EBs.

<p>A total of 4.5 μg/ml of non-pooled or pooled EBs (red symbols) were fixed to poly-L-lysine-coated plates then incubated with duplicate serial dilutions of antibodies against chlamydial outer membrane proteins: <b>(A)</b> Non-pooled EBs of serovar D with mouse anti-CT LPS, <b>(B)</b> Non-pooled EBs of serovar E with mouse anti-CT LPS <b>(C)</b> Pooled EBs of serovars D and E with mouse anti-LPS <b>(D)</b> Pooled EBs of serovars D and E with mouse anti-MOMP D <b>(E)</b> Pooled EBs of serovars D and E with mouse anti-MOMP E and <b>(F)</b> Pooled EBs of serovars D and E with mouse anti-OmcA. Antibodies against cytoplasmic antigens of pooled EBs of serovars D and E: <b>(G)</b> Rabbit anti-Euo, <b>(H)</b> Mouse anti-Hsp60, and <b>(I)</b> Rabbit anti-Scc4. 4.5 μg/ml of lysed EBs of pooled serovars D and E were evaluated with <b>(J)</b> Rabbit anti-Euo <b>(K)</b> Mouse anti-Hsp60 and <b>(L)</b> Rabbit anti-Scc4. Blue symbols represent results obtained with irrelevant isotype-matched antibodies. Mean absorbance values ± SD from one representative experiment of three are shown.</p

Poly-L-lysine coating of microtiter plates provides superior capture of EBs.

<p>Plates coated with poly-L-lysine or PBS were treated with serial dilutions of a pooled EB mixture (CT serovars D and E) in duplicate, centrifuged, and then fixed with glutaraldehyde. The plates were then washed, blocked, and incubated with a saturating concentration (5.3 μg/ml) of mouse anti-CT LPS mAb. Mean absorbance values ± SD from one representative experiment of three are presented. Poly-L-lysine or PBS-coated plates identically treated with PBS served as negative controls. The concentration of EBs shown represents the total protein in the pooled EB mixture. A concentration of 4.5 μg/ml of pooled EBs and lysed EBs was selected for use in subsequent experiments.</p

Rare coding variants in genes encoding GABAA receptors in genetic generalised epilepsies : an exome-based case-control study

Background: Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy. Methods: For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABAA receptors and was compared to the respective GABAA receptor variants of a third independent control cohort. Functional investigations were done with automated two-microelectrode voltage clamping in Xenopus laevis oocytes. Findings: Statistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABAA receptors in cases (odds ratio [OR] 2·40 [95% CI 1·41–4·10]; pNonsyn=0·0014, adjusted pNonsyn=0·019). Enrichment for these genes was validated in a whole-exome sequencing cohort of 357 sporadic and familial genetic generalised epilepsy cases and 1485 independent controls (OR 1·46 [95% CI 1·05–2·03]; pNonsyn=0·0081, adjusted pNonsyn=0·016). Comparison of genes encoding GABAA receptors in the independent replication cohort of 583 familial and sporadic genetic generalised epilepsy index cases, based on candidate-gene panel sequencing, with a third independent control cohort of 635 controls confirmed the overall enrichment of rare missense variants for 15 GABAA receptor genes in cases compared with controls (OR 1·46 [95% CI 1·02–2·08]; pNonsyn=0·013, adjusted pNonsyn=0·027). Functional studies for two selected genes (GABRB2 and GABRA5) showed significant loss-of-function effects with reduced current amplitudes in four of seven tested variants compared with wild-type receptors. Interpretation: Functionally relevant variants in genes encoding GABAA receptor subunits constitute a significant risk factor for genetic generalised epilepsy. Examination of the role of specific gene groups and pathways can disentangle the complex genetic architecture of genetic generalised epilepsy. Funding: EuroEPINOMICS (European Science Foundation through national funding organisations), Epicure and EpiPGX (Sixth Framework Programme and Seventh Framework Programme of the European Commission), Research Unit FOR2715 (German Research Foundation and Luxembourg National Research Fund)