Rapid Diagnostics of Joint Infections Using IS-Pro.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.

Enhanced Th17 responses in the appendix of children with complex compared to simple appendicitis are associated with microbial dysbiosis.

Introduction: Appendicitis is one of the most common causes of acute abdominal surgery in children. The clinical course of appendicitis ranges from simple to complex appendicitis. The mechanisms underlying the heterogeneity of appendicitis in children remain largely unclear. Dysregulated T cell responses play an important role in several inflammatory diseases of the intestine, but the extend of T cell dysregulation in appendicitis in children is less well known. Methods: To characterize appendiceal T cells in simple and complex appendicitis we performed in-depth immunophenotyping of appendiceal-derived T cells by flow cytometry and correlated this to appendiceal-derived microbiota analyses of the same patient. Results: Appendix samples of twenty children with appendicitis (n = 8 simple, n = 12 complex) were collected. T cells in complex appendicitis displayed an increased differentiated phenotype compared to simple appendicitis, including a loss of both CD27 and CD28 by CD4+ T cells and to a lesser extent by CD8+ T cells. Frequencies of phenotypic tissue-resident memory CD69+CD4+ T cells and CD69+CD8+ T cells were decreased in children with complex compared to simple appendicitis, indicating disruption of local tissue-resident immune responses. In line with the increased differentiated phenotype, cytokine production of in particular IL-17A by CD4+ T cells was increased in children with complex compared to simple appendicitis. Furthermore, frequencies of IL-17A+ CD4+ T cells correlated with a dysregulation of the appendiceal microbiota in children with complex appendicitis. Conclusion: In conclusion, disruption of local T cell responses, and enhanced pro-inflammatory Th17 responses correlating to changes in the appendiceal microbiota were observed in children with complex compared to simple appendicitis. Further studies are needed to decipher the role of a dysregulated network of microbiota and Th17 cells in the development of complex appendicitis in children.

The potential of rectal swabs to differentiate simple and complex appendicitis in children with a microbiota-based test.

Currently, accurate biomarkers differentiating simple (phlegmonous) from complex (gangrenous and/or perforated) appendicitis in children are lacking. However, both types may potentially require different treatment strategies, and the search for diagnostic modalities remains warranted. Previously, we demonstrated a distinct microbiota (both an increased bacterial diversity and abundance) in the appendix of children with complex compared to simple appendicitis. From the same cohort of patients we have collected 35 rectal swabs under general anesthesia prior to appendectomy and microbiota analysis was performed by IS-pro, a 16S-23S rDNA-based clinical microbiota profiling technique. Using the obtained IS-profiles, we performed cluster analyses (UPGMA), comparison of diversity (Shannon Diversity Index) and intensity (abundance in relative fluorescence units) on phylum level, and comparison on species level of bacteria between simple and complex appendicitis. Regarding these analyses, we observed no clear differences between simple and complex appendicitis. However, increased similarity of the microbial composition of the appendix and rectal swab was found within children with complex compared to simple appendicitis. Furthermore, PLS-DA regression analysis provided clear visual differentiation between simple and complex appendicitis, but the diagnostic power was low (highest AUC 0.65).   Conclusion: Microbiota analysis of rectal swabs may be viable to differentiate between simple and complex appendicitis prior to surgery as a supervised classification model allowed for discrimination of both types. However, the current diagnostic power was low and further validation studies are needed to assess the value of this method. What is Known: • Simple and complex appendicitis in children may require different treatment strategies, but accurate preoperative biomarkers are lacking. • Clear differentiation can be made between both types in children based upon the microbial composition in the appendix. What is New: • Increased similarity was found between the microbial composition of the appendix and rectal swab within children with complex compared to simple appendicitis. • Using a supervised classification model rectal swabs may be viable to discriminate between simple and complex appendicitis, but the diagnostic power was low.

The Vaginal Microbiota Composition and Genital Infections during and after Pregnancy among Women in Pemba Island, Tanzania.

We investigated the vaginal microbiota (VMB) composition, prevalence of genital pathogens and their association among pregnant and post-delivery women in Pemba Island, Tanzania. Vaginal swabs were collected from 90 women, at two time points during pregnancy (<20 weeks of gestational age [GA] and ≥20 weeks GA) and once after delivery, when possible. IS-pro assay was used for VMB characterization. Chlamydia trachomatis (CT), Neisseria gonorrhea (NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG) and human papillomavirus (HPV) were detected by qPCRs. VMB were mostly Lactobacillus dominant during pregnancy and non-Lactobacillus dominant post-delivery. A significant decrease in VMB richness was observed during pregnancy among paired and unpaired samples. Shannon diversity was significantly lower during pregnancy than post-delivery among unpaired samples. Klebsiella species and Streptococcus anginosus were the most commonly identified pathobionts at all timepoints. A high abundance of pathobionts was mostly seen in women with non-Lactobacillus dominant VMB. At ≥20 weeks GA timepoint during pregnancy, 63.0% of the women carrying one or more genital pathogen (either HPV, CT, TV, or MG) had L. iners dominant VMB. NG was not detected pre-delivery. This study contributes evidence on VMB composition, its changes during pregnancy and post-delivery, and their association with pathobionts and genital pathogens.

Microbiota of Children With Complex Appendicitis: Different Composition and Diversity of The Microbiota in Children With Complex Compared With Simple Appendicitis.

BACKGROUND: Two types of appendicitis are hypothesized, simple and complex, with potential different treatment strategies. To improve differentiation, underlying pathogeneses need to be further unraveled. AIM: To determine if the microbial composition in the appendix differs between children with simple and complex appendicitis. METHODS: Two-center, prospective cohort study including 40 children (0-17 years old) undergoing appendectomy for suspected appendicitis. Appendix tissue was used for IS-pro analysis to identify bacterial species by their length of 16S-23S rDNA interspacer (IS) region. Cluster analysis, based on IS-profiles, and correspondence with type of appendicitis, using Fisher exact test, was performed. Simple and complex appendicitis were compared regarding bacterial presence, intensity and diversity, using Fisher exact test and Mann-Whitney U test, respectively. RESULTS: Appendicitis was confirmed in 36 of 40 patients (16 simple, 20 complex). Cluster analysis identified 2 clusters, encompassing 34 patients. Distribution of simple and complex appendicitis was 12 (80%) and 3 (20%) versus 3 (16%) and 16 (84%) patients for clusters 1 and 2, respectively (P < 0.001). Complex appendicitis was on phylum level characterized by an increased intensity (Bacteroidetes P = 0.001, Firmicutes, Actinobacteria, Fusobacteria and Verrucomicrobia (FAFV) P = 0.005 and Proteobacteria P < 0.001) and diversity (Bacteroidetes P = 0.001 and Proteobacteria P = 0.016) and an increased abundance of 5 species (Alistipes finegoldii P = 0.009, Bacteroides fragilis P = 0.002, Escherichia coli P = 0.014, Parvimonas micra P = 0.022 and Sutterella spp P = 0.026). CONCLUSIONS: The microbial composition of the appendix differs between children with simple and complex appendicitis, regarding both composition and diversity. Future research should focus on the role of these bacteria in the pathogenesis of both types and its implications for preoperative diagnostics.

PCR Characterization of Microbiota on Contracted and Non-Contracted Breast Capsules.

BACKGROUND: The aetiology of capsular contracture around breast implants remains unclear. The leading theory is that a subclinical infection around the implant plays a role in the development of capsular contractions. Several studies found associations between the presence of bacteria and the occurrence of capsular contraction. However, it is unclear whether detected bacteria originate from the breast capsule, breast glandular tissue or skin contamination. Moreover, this has never been investigated with molecular techniques. The aim of this study was to assess the bacterial microbiota on breast capsules, glandular tissue and skin using a highly sensitive PCR assay. MATERIALS AND METHODS: Fifty breast capsules were collected during implant removal or replacement. Ten specimens of glandular breast tissue and breast skin were collected in females who were undergoing reduction mammoplasty. A sample specimen (4 mm) was sterilely obtained from all tissues. All specimens were analysed by IS-pro, a 16S-23S interspace region-based PCR assay. RESULTS: Low numbers of Staphylococcus spp. (four species in four capsules) were found on breast capsules. There was no difference in bacterial presence between normal and contracted capsules. The skin of the breast-harboured Streptococcus spp. and Staphylococcus spp. while the glandular tissue was sterile. CONCLUSION: The low numbers of bacteria found on the capsules are most likely caused by contamination during capsule removal. More and larger studies are needed to investigate the bacterial presence on breast capsules using a PCR assay. This is the first study in which breast capsules have been studied using a highly sensitive PCR assay. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

The ReceptIVFity cohort study protocol to validate the urogenital microbiome as predictor for IVF or IVF/ICSI outcome.

BACKGROUND: During the last decade, research has shown that besides the known predictive factors, such as duration of subfertility, a women's age, the body mass index, also the microbiome might affect fertility. Micro-organisms together with their genetic information and the milieu in which they interact are called the microbiome. Studies have shown that the presence of certain microbiota during assisted reproductive technology (ART) has a positive impact on the outcome. However, the potential role of using the microbiome as a predictor for outcome of ART has not yet been investigated. METHODS: In a prospective study, 300 women of reproductive age and with an indication for in-vitro Fertilization (IVF) with or without Intra Cytoplasmic Sperm Injection (ICSI) treatment will be included. Prior to the IVF or IVF-ICSI treatment, these women provided a midstream urine sample and a vaginal swab. The composition of the urinary and vaginal microbiome will be analysed with both Next Generation Sequencing and the IS-pro technique. The endpoints of the study are pregnancy achieved after fresh embryo transfer (ET) and within the subsequent year after inclusion. External validation of the findings will take place in an additional cohort of 50 women with an IVF or IVF-ICSI indication. DISCUSSION: In the proposed study, the predictive accuracy of the composition of the urinary and vaginal microbiome for IVF or IVF-ICSI outcome will be only validated for fresh ET. Follow-up has to show whether the predictive accuracy will be similar during the consecutive frozen ET's as part of the IVF or IVF-ICSI treatment or for subsequent stimulated or natural cycles. In addition, external validation will take place in another cohort and hospital. Predictive knowledge of the microbiome profile may enable couples to make a more substantiated decision on whether to continue treatment or not. Hence, the unnecessary physical and emotional burden of a failed IVF or IVF-ICSI treatment can be avoided. TRIAL REGISTRATION: ISRCTN ISRCTN83157250 . Registered 17 August 2018. Retrospectively registered.

Performance of cobas® 4800 and m2000 real-time™ assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in rectal and self-collected vaginal specimen.

A prospective, multicenter trial was designed to compare the performance characteristics of the cobas® 4800 (Roche Diagnostics, Indianapolis, IN, USA) and m2000 real-time™ (Abbott Molecular Inc., Des Plaines, IL, USA) assays for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in rectal and self-collected vaginal swabs. Rectal (n = 234) or self-collected vaginal swabs (n = 687) were obtained from consenting individuals visiting their general practitioners, dermatologists, gynecologists, sexually transmitted disease clinics, or family planning centers from May 2010 to February 2011. High concordance rates (≥96%) were observed between the cobas® 4800 and m2000 real-time™ assays for CT/NG detection in both rectal and self-collected vaginal swabs. The performance profiles confirm the usefulness of both kinds of swab types for CT and NG detection using described nucleic acid amplification tests assays. Based on this study, rectal and self-collected vaginal swabs offer a noninvasive alternative, which may improve screening for CT and NG infections.

Lymphocytic choriomeningitis virus uses a novel endocytic pathway for infectious entry via late endosomes.

The endocytic entry of lymphocytic choriomeningitis virus (LCMV) into host cells was compared to the entry of viruses known to exploit clathrin or caveolae/raft-dependent pathways. Pharmacological inhibitors, expression of pathway-specific dominant-negative constructs, and siRNA silencing of clathrin together with electron and light microscopy provided evidence that although a minority population followed a classical clathrin-mediated mechanism of entry, the majority of these enveloped RNA viruses used a novel endocytic route to late endosomes. The pathway was clathrin, dynamin-2, actin, Arf6, flotillin-1, caveolae, and lipid raft independent but required membrane cholesterol. Unaffected by perturbation of Rab5 or Rab7 and apparently without passing through Rab5/EEA1-positive early endosomes, the viruses reached late endosomes and underwent acid-induced penetration. This membrane trafficking route between the plasma membrane and late endosomes may function in the turnover of a select group of surface glycoproteins such as the dystroglycan complex, which serves as the receptor of LCMV.