Managing pregnancy of unknown location based on initial serum progesterone and serial serum hCG levels: development and validation of a two-step triage protocol.

OBJECTIVES: A uniform rationalized management protocol for pregnancies of unknown location (PUL) is lacking. We developed a two-step triage protocol to select PUL at high risk of ectopic pregnancy (EP), based on serum progesterone level at presentation (step 1) and the serum human chorionic gonadotropin (hCG) ratio, defined as the ratio of hCG at 48 h to hCG at presentation (step 2). METHODS: This was a cohort study of 2753 PUL (301 EP), involving a secondary analysis of prospectively and consecutively collected PUL data from two London-based university teaching hospitals. Using a chronological split we used 1449 PUL for development and 1304 for validation. We aimed to assign PUL as low risk with high confidence (high negative predictive value (NPV)) while classifying most EP as high risk (high sensitivity). The first triage step assigned PUL as low risk using a threshold of serum progesterone at presentation. The remaining PUL were triaged using a novel logistic regression risk model based on hCG ratio and initial serum progesterone (second step), defining low risk as an estimated EP risk of < 5%. RESULTS: On validation, initial serum progesterone ≤ 2 nmol/L (step 1) classified 16.1% PUL as low risk. Second-step classification with the risk model selected an additional 46.0% of all PUL as low risk. Overall, the two-step protocol classified 62.1% of PUL as low risk, with an NPV of 98.6% and a sensitivity of 92.0%. When the risk model was used in isolation (i.e. without the first step), 60.5% of PUL were classified as low risk with 99.1% NPV and 94.9% sensitivity. CONCLUSION: PUL can be classified efficiently into being either high or low risk for complications using a two-step protocol involving initial progesterone and hCG levels and the hCG ratio. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

A spline-based tool to assess and visualize the calibration of multiclass risk predictions.

When validating risk models (or probabilistic classifiers), calibration is often overlooked. Calibration refers to the reliability of the predicted risks, i.e. whether the predicted risks correspond to observed probabilities. In medical applications this is important because treatment decisions often rely on the estimated risk of disease. The aim of this paper is to present generic tools to assess the calibration of multiclass risk models. We describe a calibration framework based on a vector spline multinomial logistic regression model. This framework can be used to generate calibration plots and calculate the estimated calibration index (ECI) to quantify lack of calibration. We illustrate these tools in relation to risk models used to characterize ovarian tumors. The outcome of the study is the surgical stage of the tumor when relevant and the final histological outcome, which is divided into five classes: benign, borderline malignant, stage I, stage II-IV, and secondary metastatic cancer. The 5909 patients included in the study are randomly split into equally large training and test sets. We developed and tested models using the following algorithms: logistic regression, support vector machines, k nearest neighbors, random forest, naive Bayes and nearest shrunken centroids. Multiclass calibration plots are interesting as an approach to visualizing the reliability of predicted risks. The ECI is a convenient tool for comparing models, but is less informative and interpretable than calibration plots. In our case study, logistic regression and random forest showed the highest degree of calibration, and the naive Bayes the lowest.

Rationalizing the management of pregnancies of unknown location: temporal and external validation of a risk prediction model on 1962 pregnancies.

STUDY QUESTION: Can we accurately define a group of pregnancies of unknown location (PULs) as low risk in order to safely reduce follow-up for these pregnancies and allocate resources to pregnancies at an increased risk of being ectopic? SUMMARY ANSWER: Prediction model M4 classified around 70% of PULs as low risk, of which around 97% were later characterized as failed PULs or intrauterine pregnancies (IUPs), while still classifying 88% of ectopic pregnancies as high risk. WHAT IS KNOWN ALREADY: Depending on the level of suspicion of ectopic pregnancy (EP), women with a PUL receive a lengthy follow-up in order to confirm the location and viability of the pregnancy. STUDY DESIGN, SIZE, DURATION: A multi-centre diagnostic accuracy study of 1962 patients was carried out between 2003 and 2007 for retrospective temporal validation and between 2009 and 2011 for prospective external validation. The reference standard is the final characterization of PUL as failed pregnancies or IUPs (low risk), or as ectopic pregnancies (high risk). M4 is a multinomial logistic regression model based on the serum human chorionic gonadotrophin (hCG) levels at presentation and 48 h later. PARTICIPANTS/MATERIALS, SETTING, METHODS: Temporal validation data from 1341 PULs collected at St George's Hospital in London were available, of which 53% were failed, 39% were intrauterine and 8% were ectopic pregnancies. External validation data from 621 PULs collected at four other London-based teaching hospitals were available, of which 63% were failed, 22% were intrauterine and 15% were ectopic pregnancies. MAIN RESULTS AND THE ROLE OF CHANCE: The EP rate varied between 8 and 16% across the five hospitals. At St George's, 980 [73.1%, 95% confidence interval (CI): 70.5-75.4] PULs were considered low risk. Of these, 963 were failed PULs or IUPs (98.3%, 95% CI: 97.2-98.9) and 17 were ectopic pregnancies. At the other four hospitals, 62-75% were considered low risk, with 96-98% of these turning out to be failed PUL or IUP. Eighty-five percent (95% CI: 76.8-90.2) of the ectopic pregnancies were considered high risk at St George's, compared with 80-92% in the other hospitals. LIMITATIONS, REASONS FOR CAUTION: Of total, 120 patients had been excluded due to loss to follow-up, and a further 102 patients because of missing hCG levels due to differences in local clinical practice. There are variations in the definition of a PUL used in different countries. WIDER IMPLICATIONS OF THE FINDINGS: The suggested protocol could safely reduce the follow-up in the majority of PUL such that units could increase the focus on women at a risk of complications. This would lead to a change in the management of the majority of women with a PUL and a more efficient use of resources. At the end of the manuscript, we provide a link to enable clinicians to use the protocol. STUDY FUNDING/COMPETING INTEREST(S): B.V.C. is supported by a postdoctoral fellowship from the Research Foundation Flanders (FWO). K.V.H. is supported by a fellowship from the Flanders' Agency for Innovation by Science and Technology (IWT-Vlaanderen), by the Research Council KU Leuven (GOA MaNet), by the Flemish Government (iMinds) and by the Belgian Federal Science Policy Office (IUAP P7/DYSCO). T.B. is supported by the Imperial Healthcare NHS Trust NIHR Biomedical Research Centre. No competing interests are declared.

Validation of MALDI-TOF MS for rapid classification and identification of lactic acid bacteria, with a focus on isolates from traditional fermented foods in Northern Vietnam.

AIMS: To evaluate the potential use of MALDI-TOF MS for fast and reliable classification and identification of lactic acid bacteria (LAB) from traditional fermented foods. METHODS AND RESULTS: A total of 119 strains of LAB from fermented meat (nem chua) were analysed with both (GTG)(5)-PCR fingerprinting and MALDI-TOF MS. Cluster analysis of the profiles revealed five species represented by a single isolate both in (GTG)(5)-PCR and in MALDI-TOF MS; five species grouped alike for (GTG)(5)-PCR and for MALDI-TOF MS; however, differences in minimal similarity between the delineated (GTG)(5)-PCR and MALDI-TOF MS clusters could be observed; three species showed more heterogeneity in their MALDI-TOF MS profiles compared to their (GTG)(5)-PCR profiles; two species, each represented by a single MALDI-TOF cluster, were subdivided in the corresponding (GTG)(5)-PCR dendrogram. As proof of the identification potential of MALDI-TOF MS, LAB diversity from one fermented mustard sample was analysed using MALDI-TOF MS. PheS gene sequencing was used for validation. CONCLUSIONS: MALDI-TOF MS is a powerful, fast, reliable and cost-effective technique for the identification of LAB associated with the production of fermented foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Food LAB can be identified using MALDI-TOF MS, and its application could possibly be extended to other food matrices and/or other food-derived micro-organisms.

Lobular and non-lobular breast cancers differ regarding axillary lymph node metastasis: a cross-sectional study on 4,292 consecutive patients.

Invasive lobular carcinoma (ILC) accounts for 8-14% of all breast cancers and carries distinct prognostic and biologic implications. The goal of our study was to investigate the impact of lobular histology on axillary lymph node (ALN) involvement. This is a cross-sectional study of 4,292 consecutive patients surgically treated for breast carcinoma at the University Hospitals Leuven. Logistic regression analysis was used to relate ILC to lymph node involvement while controlling for the following clinicopathologic features: tumor size, multifocal disease, tumor grade, lobular subtype and the combined steroid, and Her-2 status. Odds ratios (ORs) and 95% confidence intervals (CIS) were computed. A subgroup analysis was performed for patients that underwent a sentinel lymph node (SLN) procedure. The observed incidence of ILC was 13%. ILCs were larger, were more often grade II, multifocal, steroid receptor positive and Her-2 negative, and tended to be present in older patients. Incidence of ALN involvement was 42.0% for ILCs versus 38.3% for other tumors (OR 1.17, 95% CI 0.97-1.40). For the SLN subgroup, ILCs were less often ALN positive than non-ILCs (20.5% versus 28.3%, OR 0.66, 95% CI: 0.41-1.00). In the multivariable analysis, the lobular subtype was identified as less likely to have ALN involvement (adjusted OR 0.66, 95% CI 0.53-0.82). The analysis for the SLN subgroup showed comparable results (adjusted OR 0.49, 95% CI 0.30-0.78). This study has demonstrated that the lobular subtype is an independent predictor of lymph node involvement with ILC having a lower incidence of involved lymph nodes. The mildly higher incidence of ALN metastasis in lobular cancers in univariable analysis is not due to the lobular subtype, but due to confounding factors that interact with lymph node involvement.

Use of next-generation sequencing in microbial risk assessment.

Despite the ever increase in rigorous control and monitoring measures to assure safe food along the entire farm-to-fork chain, the past decade has also witnessed an increase in microbial food alerts. Hence, research on food safety and quality remain of utmost importance. Complementary, and at least as important, is the necessity to be able to assess the potential microbial risks along the food chain. Risk assessment relies on sound scientific data. Unfortunately, often, quality data are limited if not lacking. High-throughput tools such as next-generation sequencing (NGS) could fill this gap. NGS approaches can be used to generate ample qualitative and quantitative data to be used in the risk assessment process. NGS applications are not new in food microbiology with applications ranging from pathogen detection along the food chain, food epidemiology studies, whole genome analysis of food-associated microorganisms up to describing complete food microbiomes. Yet, its application in the area of microbial risk assessment is still at an early stage and faces important challenges. The possibilities of NGS for risk assessment are ample, but so are the questions on the subject. One of the major strengths of NGS lies in its capacity to generate a lot of data, but to what extend can this wealth be of use in hazard identification, hazard characterisation and exposure assessment to perform a sound risk characterisation, which in turn will make it possible to take substantiated risk management decisions.

In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium.

Next to health promoting effects, the functional aspect of probiotic strains also involves their capacity to reach the colon as viable metabolically active cells. The present study aimed to assess the potential of 24 probiotic product isolates and 42 human reference strains of Bifidobacterium to survive gastrointestinal transit under in vitro conditions. The survival capacity of exponential and stationary phase cultures upon exposure to gastric and small intestinal juices was determined using a recently developed microplate-based assay in combination with the LIVE/DEAD BacLight Bacterial Viability kit. All 66 strains tested displayed a considerable loss in viability during exposure to an acidic pepsin containing solution (pH 2.0). Among the 10 taxa tested, cultures of B. animalis ssp. lactis appeared to be most capable to survive gastric transit. Although to a lesser extent, the presence of bile salts also affected the viability of most of the strains tested. Except for 3 strains, all 66 strains showed bile salt hydrolase activity using an agar-based assay. In contrast, the bifidobacterial strains used in this study appeared to possess a natural ability to survive the presence of pancreatin (pH 8.0). Although the effect was not significant, a slightly enhanced tolerance to gastrointestinal transit was observed when cells were in the stationary phase, especially when exposed to acid, compared with cells being in the exponential phase. Survival in the gastrointestinal tract appeared to be largely strain-dependent and hence implies that different strains will likely display a different behavior in functionality. The assay used in this study allows an initial assessment of strains for use as probiotic cultures prior to selecting potential candidate strains for further investigation in vivo.

Selected Lactobacillus strains isolated from sugary and milk kefir reduce Salmonella infection of epithelial cells in vitro.

The isolation of potentially probiotic strains and the subsequent study of their properties are very important steps to gain insight in the health benefits ascribed to sugary and milk kefir. The aim of the present study was to characterise fifteen Lactobacillus strains isolated from these beverages by determining some surface properties and their ability to antagonise enterocyte cell damage after Salmonella infection in vitro. Lactobacillus surface properties were determined by hydrophobicity, autoaggregation, and coaggregation assays with Salmonella. In addition, lactobacilli adhesion to Caco-2/TC-7 cells and the effect on Salmonella invasion were evaluated. Finally, the disassembly of F-actin cytoskeleton on intestinal epithelial cells was assayed in vitro when Salmonella infection was performed in the presence of selected Lactobacillus strains. Ten out of the 15 strains showed a high adhesion capacity to Caco-2/TC-7 cells. Most of the strains were hydrophilic and non-autoaggregating. Strains isolated from sugary kefir were non-coaggregating with Salmonella, while strains Lactobacillus paracasei CIDCA 83120, 83121, 83123, 83124, 8339, 83102 isolated from milk kefir were able to coaggregate after 1 h. L. paracasei CIDCA 8339 and Lactobacillus kefiri CIDCA 83102 were able to diminish Salmonella invasion to the enterocytes. An antagonistic effect on cytoskeleton disruption elicited by the pathogen was also demonstrated. Our results suggest that both strains isolated from milk kefir could be considered as appropriate probiotic candidates.

Practical guidance for applying the ADNEX model from the IOTA group to discriminate between different subtypes of adnexal tumors.

All gynecologists are faced with ovarian tumors on a regular basis, and the accurate preoperative diagnosis of these masses is important because appropriate management depends on the type of tumor. Recently, the International Ovarian Tumor Analysis (IOTA) consortium published the Assessment of Different NEoplasias in the adneXa (ADNEX) model, the first risk model that differentiates between benign and four types of malignant ovarian tumors: borderline, stage I cancer, stage II-IV cancer, and secondary metastatic cancer. This approach is novel compared to existing tools that only differentiate between benign and malignant tumors, and therefore questions may arise on how ADNEX can be used in clinical practice. In the present paper, we first provide an in-depth discussion about the predictors used in ADNEX and the ability for risk prediction with different tumor histologies. Furthermore, we formulate suggestions about the selection and interpretation of risk cut-offs for patient stratification and choice of appropriate clinical management. This is illustrated with a few example patients. We cannot propose a generally applicable algorithm with fixed cut-offs, because (as with any risk model) this depends on the specific clinical setting in which the model will be used. Nevertheless, this paper provides a guidance on how the ADNEX model may be adopted into clinical practice.

Antimicrobial susceptibility of Bifidobacterium strains from humans, animals and probiotic products.

OBJECTIVES: The aim of this study was to assess the antimicrobial susceptibility of a taxonomically diverse set of Bifidobacterium strains to different classes of antimicrobial agents using a recently described medium. METHODS: The susceptibility of 100 strains encompassing 11 bifidobacterial species originating from humans, animals and probiotic products to 12 antimicrobial agents was tested by agar overlay disc diffusion. Based on these results, one or two strains per species were selected for susceptibility testing to nine antibiotics by broth microdilution using the Lactic acid bacteria Susceptibility test Medium (LSM) supplemented with cysteine. The genotypic basis of atypical tetracycline resistance was further characterized using PCR, Southern blotting and partial sequencing. RESULTS: Based on the distribution of inhibition zone diameters and MIC values, all strains tested were susceptible to amoxicillin, chloramphenicol, erythromycin, quinupristin/dalfopristin, rifampicin and vancomycin. Our data also reinforce earlier observations indicating that bifidobacteria are intrinsically resistant to gentamicin, sulfamethoxazole and polymyxin B. Susceptibility to trimethoprim, trimethoprim/sulfamethoxazole, ciprofloxacin, clindamycin, tetracycline and minocycline was variable. The tet(W) gene was responsible for tetracycline resistance in 15 strains including 7 probiotic isolates belonging to the taxa Bifidobacterium animalis subsp. lactis and Bifidobacterium bifidum. This gene was present in a single copy on the chromosome and did not appear to be associated with the conjugative transposon TnB1230 previously found in tet(W)-containing Butyrivibrio fibrisolvens. CONCLUSIONS: The use of the LSM + cysteine medium allowed us to discriminate between intrinsic and atypical resistance properties of bifidobacteria and sets the scene for future definition of epidemiological cut-off values for all important Bifidobacterium species. The presence of an acquired tet(W) gene in several probiotic product isolates stresses the need for a minimal safety evaluation during the selection of Bifidobacterium strains for probiotic use.