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.

The effect of selective decontamination on the intestinal microbiota as measured with IS-pro: a taxonomic classification tool applicable for direct evaluation of intestinal microbiota in clinical routine.

Selective decontamination of the digestive tract (SDD) is aimed at elimination of potential pathogenic microorganisms. In this study, the effect of SDD on gut microbiota was evaluated in a large homogenous group of elective colorectal cancer surgery patients. Rectal swabs were taken from 118 patients undergoing colorectal surgery. These patients were randomly assigned to receive perioperative SDD or to the control group (no SDD). Rectal swabs were taken prior to surgery, 3 days after commencing administration of SDD. Gut microbial profiles were obtained with the IS-pro technique, a standardized microbiota profiling assay applicable in clinical routine. Differences in abundance for different taxonomical groups and diversity between the groups were assessed. Unsupervised and supervised classification techniques were used to assess microbial signatures, differentiating between the SDD group and the control group. Patients in the SDD group had different gut microbial signatures than in the control group, also in phyla that are not a target for SDD. Escherichia coli, Sutterella spp., Faecalibacterium prausnitzii, and Streptococcus spp. were the species that differed the most between the two groups. The SDD group showed clustering into two subgroups. In one subgroup, a decrease in Proteobacteria was observed, whereas the other subgroup showed a shift in Proteobacteria species. This study shows that SDD not only decreases colonization of the gastrointestinal tract with potential pathogenic Gram-negative microorganisms, but also reduces the abundance of normal colonizers of our gastrointestinal system and leads to a shift in total microbiota composition.

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 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 influence of age, gender and race/ethnicity on the composition of the human axillary microbiome.

OBJECTIVE: The microbial community plays an important role in the generation of human axillary odour by transforming odourless natural secretions into volatile odorous molecules. A limited number of traditional culturing methods and molecular based research have been performed to characterize the human axillary microbiome in small collection sample sizes. Moreover, only a few have considered the interpersonal variations across age, gender or race/ethnicity, and none have included all three variables within one single study. The aim of this study was to characterize the axillary microbiome of healthy subjects across different age groups, genders and races/ethnicities in a large sample size. METHODS: The underarm skin swab samples were collected from 169 healthy subjects. The axillary microbiome was analysed by IS-pro, a clinically validated high-throughput DNA fingerprinting technique. RESULTS: The results indicate that the senior subjects (55+) tend to have a higher number of total bacterial than younger adults (of a defined age). The diversity of odour causing bacteria, e.g. corynebacteria, increases with age. Among the three races/ethnicities studied, East Asians have a unique microbial composition compared to Caucasians and Hispanics, which may contribute to the different odour profiles observed among the races/ethnicities studied. CONCLUSION: Human axillary microbiome varies by age, gender and race/ethnicity. This study has provided an unprecedented fundamental knowledge about the axillary microbiota as a function of age, gender and race/ethnicity.

OBJECTIF: La population microbienne joue un rôle important dans la génération de l'odeur axillaire par la transformation de sécrétions naturelles inodores en molécules odorantes et volatiles. Un nombre limité d'études par culture traditionnelle et de recherches moléculaires ont été réalisées pour caractériser le microbiome axillaire humain dans des échantillons de prélèvements de petite taille. En outre, seules quelques-unes de ces études ont tenu compte des variations interpersonnelles à travers l'âge, le sexe ou la race/l'origine ethnique, et aucune n'a inclus les trois variables dans une seule et même recherche. Le but de cette étude est de caractériser le microbiome de sujets sains est de réunir différents groupes d'âge, sexes et races/origines ethniques dans un échantillon important. MÉTHODES: Les échantillons de frottis cutanés de l'aisselle ont été recueillis sur 169 sujets sains. Le microbiome axillaire a été analysé par ISpro, une technique cliniquement validée d'empreinte ADN à haut débit. RÉSULTATS: Les résultats indiquent que les sujets séniors (55 ans et plus) ont tendance à présenter un plus grand nombre de bactéries que les adultes plus jeunes (d'un âge défini). La diversité des bactéries odorantes, par exemple, de type corynebacterium, augmente avec l'âge. Parmi les trois races/origines ethniques étudiées, les populations asiatiques présentent une composition microbienne unique par rapport aux populations caucasiennes et hispaniques, ce qui pourrait contribuer aux différents profils d'odeur observés dans les races/origines ethniques prises en compte. CONCLUSION: Le microbiome axillaire varie selon l'âge, le sexe et la race/l'origine ethnique. Cette étude fournit une connaissance fondamentale sans précédent sur la flore axillaire en fonction de l'âge, du sexe et de la race/l'origine ethnique.

Interpretation of microbiota-based diagnostics by explaining individual classifier decisions.

BACKGROUND: The human microbiota is associated with various disease states and holds a great promise for non-invasive diagnostics. However, microbiota data is challenging for traditional diagnostic approaches: It is high-dimensional, sparse and comprises of high inter-personal variation. State of the art machine learning tools are therefore needed to achieve this goal. While these tools have the ability to learn from complex data and interpret patterns therein that cannot be identified by humans, they often operate as black boxes, offering no insight into their decision-making process. In most cases, it is difficult to represent the learning of a classifier in a comprehensible way, which makes them prone to be mistrusted, or even misused, in a clinical environment. In this study, we aim to elucidate microbiota-based classifier decisions in a biologically meaningful context to allow their interpretation. RESULTS: We applied a method for explanation of classifier decisions on two microbiota datasets of increasing complexity: gut versus skin microbiota samples, and inflammatory bowel disease versus healthy gut microbiota samples. The algorithm simulates bacterial species as being unknown to a pre-trained classifier, and measures its effect on the outcome. Consequently, each patient is assigned a unique quantitative estimation of which species in their microbiota defined the classification of their sample. The algorithm was able to explain the classifier decisions well, demonstrated by our validation method, and the explanations were biologically consistent with recent microbiota findings. CONCLUSIONS: Application of a method for explaining individual classifier decisions for complex microbiota analysis proved feasible and opens perspectives on personalized therapy. Providing an explanation to support a microbiota-based diagnosis could guide decisions of clinical microbiologists, and has the potential to increase their confidence in the outcome of such decision support systems. This may facilitate the development of new diagnostic applications.

Robust Microbiota-Based Diagnostics for Inflammatory Bowel Disease.

Strong evidence suggests that the gut microbiota is altered in inflammatory bowel disease (IBD), indicating its potential role in noninvasive diagnostics. However, no clinical applications are currently used for routine patient care. The main obstacle to implementing a gut microbiota test for IBD is the lack of standardization, which leads to high interlaboratory variation. We studied the between-hospital and between-platform batch effects and their effects on predictive accuracy for IBD. Fecal samples from 91 pediatric IBD patients and 58 healthy children were collected. IS-pro, a standardized technique designed for routine microbiota profiling in clinical settings, was used for microbiota composition characterization. Additionally, a large synthetic data set was used to simulate various perturbations and study their effects on the accuracy of different classifiers. Perturbations were validated in two replicate data sets, one processed in another laboratory and the other with a different analysis platform. The type of perturbation determined its effect on predictive accuracy. Real-life perturbations induced by between-platform variation were significantly greater than those caused by between-laboratory variation. Random forest was found to be robust to both simulated and observed perturbations, even when these perturbations had a dramatic effect on other classifiers. It achieved high accuracy both when cross-validated within the same data set and when using data sets analyzed in different laboratories. Robust clinical predictions based on the gut microbiota can be performed even when samples are processed in different hospitals. This study contributes to the effort to develop a universal IBD test that would enable simple diagnostics and disease activity monitoring.

Composition and stability of intestinal microbiota of healthy children within a Dutch population.

Numerous diseases linked to microbial imbalance can be traced back to childhood, illustrating the impact of the juvenile microbiota development from infancy toward adulthood. However, knowledge on this subject is currently very limited. The primary aim of this study was to characterize composition and short- and long-term stability of the intestinal microbiota in healthy children. Between November 2011 and June 2014, 61 children 2 to 18 yr of age from different areas in The Netherlands were included and instructed to collect fecal samples weekly, for 6 wk, and a follow-up sample after 18 mo. The intergenic spacer profiling technique (IS-pro) was used to analyze all available fecal samples. Microbial diversity was calculated by the Shannon diversity index and individual compositional stability by comparing all collection time points. Microbial stability varied per phylum (P< 0.0005), declined rapidly in a short time period, and subsequently stabilized on the long run with very gradual variation, leading to an overall compositional stability of 70% on average over a period of 18 mo. Higher species diversity was correlated to a higher overall compositional stability (P< 0.001). We observed an age-independent bacterial shared core consisting of a limited number of species. In conclusion, in this study, we showed that microbial composition stability in children varied per phylum, at both short-term and long-term intervals. Healthy children seem to share a microbiome core consisting of a limited number of species.-De Meij, T. G. J., Budding, A. E., de Groot, E. F. J., Jansen, F. M., Kneepkens, C. M. F., Benninga, M. A., Penders, J., van Bodegraven, A. A., Savelkoul, P. H. M. Composition and stability of intestinal microbiota of healthy children within a Dutch population.

Clinical Applicability of Microbiota Sampling in a Subfertile Population: Urine versus Vagina.

The urogenital microbiota is increasingly gaining recognition as a significant contributor to reproductive health. Recent studies suggest that microbiota can serve as predictors for fertility treatment outcomes. Our objective was to investigate the degree of similarity in microbial composition between patient-collected urine and vaginal samples in a subfertile population. We enrolled women of reproductive age (20-44 years) diagnosed with subfertility and requiring in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (IVF-ICSI) treatment. They self-collected both mid-stream urine samples and vaginal swabs before commencing the IVF or IVF-ICSI procedure. All samples were analysed using the intergenic spacer profiling (IS-pro) technique, a rapid clinical microbiota analysis tool. The main outcome measures were the degree of similarity of microbial composition between the two different, but simultaneously collected, samples. Our findings revealed a high correlation (R squared of 0.78) in microbiota profiles between paired urine and vaginal samples from individual patients. Nevertheless, the urinary microbiota profiles contained fewer species compared to the vaginal microbiota, resulting in minor but distinguishable differences. Furthermore, different subfertility diagnoses appeared to be associated with differences in microbial profiles. A noteworthy observation was the exclusive presence of Escherichia coli (E. coli) in both samples of women diagnosed with male factor subfertility. In conclusion, since urinary microbiota profiles seem to represent a diluted version of the vaginal microbiota, vaginal microbiome sampling to predict fertility treatment outcome seems preferable. To enhance the success of fertility treatments, further research is needed to gain deeper insights into a putative causal role of microbiota in the mechanisms of subfertility.

Potential of Molecular Culture in Early Onset Neonatal Sepsis Diagnosis: A Proof of Principle Study.

Delay in the time-to-positivity of a peripheral blood culture (PBC), the gold standard for early onset neonatal sepsis (EOS) diagnosis, has resulted in excessive use of antibiotics. In this study, we evaluate the potential of the rapid Molecular Culture (MC) assay for quick EOS diagnosis. In the first part of this study, known positive and spiked blood samples were used to assess the performance of MC. In the in vivo clinical study, the second part of this study, all infants receiving antibiotics for suspicion of EOS were included. At initial EOS suspicion, a blood sample was collected for PBC and MC. MC was able to detect bacteria present in the spiked samples even when the bacterial load was low. In the clinical study, MC was positive in one infant with clinical EOS (Enterococcus faecalis) that was not detected by PBC. Additionally, MC was positive in two infants without clinical sepsis (Streptococcus mitis and multiple species), referred to as contamination. The other 37 samples were negative both by MC and PBC. MC seems to be able to detect bacteria even when the bacterial load is low. The majority of MC and PBC results were comparable and the risk for contamination and false positive MC results seems to be limited. Since MC can generate results within 4 h following sampling compared with 36-72 h in PBC, MC may have the potential to replace conventional PBC in EOS diagnostics in order to guide clinicians on when to discontinue antibiotic therapy several hours after birth.

Capnocytophaga canimorsus Mycotic Aortic Aneurysm After a Dog Bite.

Introduction: Mycotic aortic aneurysm is defined as dilatation of the aortic wall due to infection caused by a variety of microorganisms and is associated with high mortality rates. This case report describes a patient with a rapid growing mycotic infrarenal aneurysm caused by Capnocytophaga canimorsus following a dog bite. Report: A 61 year old male professional dog handler presented with a history of progressive abdominal pain and constitutional symptoms. He had been bitten by a Pit Bull Terrier dog that was attacking a young girl three weeks prior to the onset of complaints. Investigations revealed a mycotic infrarenal aortic aneurysm that grew 0.5 cm in only three days. Open surgical repair consisting of an infrarenal aorto-aortic bypass with a 21 mm × 15 cm bovine bioprosthesis was performed successfully. All cultures and biopsies were negative and the subsequent 16S-23S rRNA intergenic spacer region based polymerase chain reaction (IS-pro) technique revealed C. canimorsus, a Gram negative bacterial pathogen that lives as a commensal in the gingival flora of dogs and cats that can cause a variety of severe infections, as the causative agent. Identification made it possible to treat the patient with eight weeks of intravenous followed by four weeks of oral antibiotics. At the last follow up over a year after surgery, the patient was symptom free, without infection and on ultrasound examination there were no signs of complications or aneurysm formation. Discussion: This case highlights C. canimorsus as a rare cause of a rapid growing mycotic aortic aneurysm following a dog bite. 16S-23S rRNA profiling (IS-pro) led to the identification of the bacterial pathogen. The use of biological grafts should be considered in the management of mycotic aortic aneurysms.

Effects of Antibiotics on the Intestinal Microbiota of Mice.

Studies on human and mouse gastrointestinal microbiota have correlated the composition of the microbiota to a variety of diseases, as well as proved it vital to prevent colonization with resistant bacteria, a phenomenon known as colonization resistance. Antibiotics dramatically modify the gut community and there are examples of how antibiotic usage lead to colonization with resistant bacteria [e.g., dicloxacillin usage selecting for ESBL-producing E. coli carriage], as shown by Hertz et al. Here, we investigated the impact of five antibiotics [cefotaxime, cefuroxime, dicloxacillin, clindamycin, and ciprofloxacin] on the intestinal microbiota in mice. Five different antibiotics were each given to groups of five mice. The intestinal microbiotas were profiled by use of the IS-pro analysis; a 16S-23S rDNA interspace [IS]-region-based profiling method. For the mice receiving dicloxacillin and clindamycin, we observed dramatic shifts in dominating phyla from day 1 to day 5. Of note, diversity increased, but overall bacterial load decreased. For ciprofloxacin, cefotaxime, and cefuroxime there were few overall changes. We speculate that antibiotics with efficacy against the abundant anaerobes in the gut, particularly Bacteroidetes, can in fact be selected for resistant bacteria, disregarding the spectrum of activity.

Microbiome on the Bone-Anchored Hearing System: A Prospective Study.

The bone-anchored hearing system (BAHS) has evolved to a common treatment option for various types of hearing revalidation. The BAHS consists of an implant in the skull that breeches the skin. Soft tissue reactions are a common complication associated with BAHS and are generally poorly understood. This study aims to investigate the influence of BAHS and associated skin reactions around the implant. A total of 45 patients were prospectively followed from implantation up to at least 1 year. Swabs were obtained at baseline, 12 weeks follow-up and during cases of inflammation (Holgers score ≥2). The microbiota was assessed using IS-proTM, a bacterial profiling method based on the interspace region between the 16S-23S rRNA genes. Detection of operational taxonomic units, the Shannon Diversity Index, sample similarity analyses and Partial Least Squares Discriminant Analysis (PLS-DA) were employed. Staphylococcus epidermidis, Streptococcus pneumoniae/mitis, Propionibacterium acnes, Staphylococcus capitis, Staphylococcus hominis, Bifidobacterium longum, Haemophilus parainfluenzae, Lactobacillus rhamnosus, Bordetella spp., Streptococcus sanguinis, Peptostreptococcus anaerobius, Staphylococcus aureus, Lactococcus lactis, Enterobacter cloacae, and Citrobacter koseri were the most commonly found bacterial species. S. pneumoniae/mitis was significantly more often observed after implantation, whereas P. acnes was significantly less observed after implantation compared with baseline. The relative abundance of S. epidermidis (17%) and S. aureus (19.4%) was the highest for the group of patients with inflammation. The Shannon Diversity Index was significantly increased after implantation compared with pre-surgical swabs for Firmicutes, Actinobacteria, Fusobacteria, Verrucomicrobia (FAFV), but not for other phyla. When combining all phyla, there was no significant increase in the Shannon Diversity Index. The diversity index was similar post-surgically for patients experiencing inflammation and for patients without inflammation. With a supervised classifier (PLS-DA), patients prone to inflammation could be identified at baseline with an accuracy of 91.7%. In addition, PLS-DA could classify post-surgical abutments as non-inflamed or inflamed with an accuracy of 97.7%. This study shows the potential of using IS-proTM to describe and quantify the microbiota associated with the percutaneous BAHS. Furthermore, the results indicate the possibility of an early identification of patients susceptible to adverse skin reaction following implantation. Both S. aureus and S. epidermidis should be considered as relevant bacteria for BAHS-associated inflammation.

Ulcerative Gastritis and Esophagitis in Two Children with Sarcina ventriculi Infection.

Sarcina ventriculi is a Gram-positive, obligate anaerobic coccus, with a characteristic morphology. Only 22 cases of human infections by this microorganism, including 7 in children, have been reported in literature so far. Affected subjects usually present with abdominal pain, nausea, vomiting, and delayed gastric emptying. However, life-threatening complications, like emphysematous gastritis and gastric perforation have also been described. Gastroparesis and gastric outlet obstruction have been considered as a potential etiologic factor. All pediatric cases described thus far presented with concomitant gastrointestinal pathology, such as Helicobacter pylori gastritis, celiac disease, infection with Giardia lamblia or Candida spp. Here, we report two children with S. ventriculi infection, in whom the diagnosis was established by typical histological findings in mucosal biopsies. The first child presented with hematemesis due to ulcerative esophagitis and gastritis, the second child with a history of esophageal stricture had ulcerative gastritis. Confirmation of S. ventriculi infection is feasible by molecular microbiota detection methods, since this microorganism cannot be detected by classical culture techniques. Prompt treatment with antibiotics could prevent life-threatening complications.