Elisabeth M. Terveer

Comparative analysis of an expanded Clostridium difficile reference strain collection reveals genetic diversity and evolution through six lineages

Clostridium difficile is an anaerobic bacillus that resides in the gut and has rapidly emerged as a leading cause of antibiotic associated diarrheal disease in humans. The genetic basis of the pathogenicity of C. difficile remains poorly understood. In this study we aimed at characterizing the genetic diversity of C. difficile strains by three different methods (PCR ribotyping, multilocus sequence typing and genetic markers) to improve the typing of C. difficile. Our study was performed on a reference collection (Leeds-Leiden/ECDC) of C. difficile PCR ribotype (RT) strains (n=70) expanded with six PCR RT strains highly related to the emerging PCR RTs 027 and 078. Besides PCR ribotyping we used multilocus sequence typing (MLST) using seven housekeeping genes (MLST 7HG) that has recently been developed for characterizing C. difficile isolates as well as analysis of unique genetic markers. Evolutionary relatedness of the sequences determined by MLST 7HG was analyzed in phylogenetic analysis. In total 56 MLST 7HG sequence types (STs) were identified, nine of which were new. Phylogeny reconstruction of the reference set of strains supplemented with the online available C. difficile MLST reference database, revealed six monophyletic lineages of closely related STs. ST-122 (PCR RT131) formed a well-separated branch in the tree and was thus designated as a novel lineage. Furthermore, we confirmed that several PCR RTs are highly related to the emerging PCR RTs 027 and 078 since these types display the same STs (ST-1 and ST-11, respectively). Based on the observed results, we conclude that MLST 7HG is a valuable method to study C. difficile phylogeny.

Oral polio vaccine influences the immune response to BCG vaccination. A natural experiment

Background Oral polio vaccine (OPV) is recommended to be given at birth together with BCG vaccine. While we were conducting two trials including low-birth-weight (LBW) and normal-birth-weight (NBW) infants in Guinea-Bissau, OPV was not available during some periods and therefore some infants did not receive OPV at birth, but only BCG. We investigated the effect of OPV given simultaneously with BCG at birth on the immune response to BCG vaccine. Methods and Findings We compared the in vitro and the in vivo response to PPD in the infants who received OPV and BCG with that of infants who received BCG only. At age 6 weeks, the in vitro cytokine response to purified protein derivate (PPD) of M. Tuberculosis was reduced in LBW and NBW infants who had received OPV with BCG. In a pooled analysis receiving OPV with BCG at birth was associated with significantly lower IL-13 (p = 0.041) and IFN-γ (p = 0.004) and a tendency for lower IL-10 (p = 0.054) in response to PPD. Furthermore, OPV was associated with reduced in vivo response to PPD at age 2 months, the prevalence ratio (PR) of having a PPD reaction being 0.75 (0.58–0.98), p = 0.033, and with a tendency for reduced likelihood of having a BCG scar (0.95 (0.91–1.00), p = 0.057)). Among children with a scar, OPV was associated with reduced scar size, the regression coefficient being −0.24 (−0.43—0.05), p = 0.012. Conclusions This study is the first to address the consequences for the immune response to BCG of simultaneous administration with OPV. Worryingly, the results indicate that the common practice in low-income countries of administering OPV together with BCG at birth may down-regulate the response to BCG vaccine.

Virtual reality in laparoscopic skills training: Is haptic feedback replaceable?

Abstract With the emphasis on laparoscopic skills training outside of the operating room (OR), simulators are constantly being developed and improved. Virtual reality (VR) trainers have been looking for solutions to compensate their lack of haptic feedback. A possible solution is the addition of kinematic interaction between laparoscopic instruments and objects. The aim of the study was to determine whether this interaction can replace haptic feedback that is naturally present in box trainers. Novices (n = 50) were randomly assigned to training in a conventional VR setup (VR-I), a VR environment with additional kinematic interaction (VR-II), a box trainer equivalent of these setups (Box-I or Box-II), or to a control group. An identical cylinder task was performed in all four training setups. The effect was established by comparing the performance before and after training during a tissue handling task, using Wilcoxon signed-rank tests. The controls did not improve significantly. The VR-I group improved in time, whereas VR-II and both box trainer groups improved in time, path length and motion in depth. With respect to haptic feedback, box training models are superior to VR systems. However, additional kinematic interaction between instruments and objects can be a promising surrogate for haptic feedback in VR systems.

Prevalence of colistin resistance gene (mcr-1) containing Enterobacteriaceae in feces of patients attending a tertiary care hospital and detection of a mcr-1 containing, colistin susceptible E. coli

The emergence of the plasmid-mediated mcr colistin resistance gene in the community poses a potential threat for treatment of patients, especially when hospitalized. The aim of this study was to determine the prevalence of all currently known mcr mediated colistin resistance gene in fecal samples of patients attending a tertiary care hospital. From November 2014 until July 2015, fecal samples of patients attending the Leiden University Medical Center were collected and screened for presence of mcr using real-time PCR. Two of 576 patients were positive for mcr-1, resulting in a prevalence of 0.35%, whereas no mcr-2 was found. One of these samples was culture negative, the second sample contained a blaCMY-2 and mcr-1 containing E.coli. This strain belonged to Sequence Type 359 and serotype O177:H21. The mcr-1 containing E.coli was phenotypically susceptible to colistin with a MIC of ≤ 0.25mg/l, due to a 1329bp transposon IS10R inserted into the mcr-1 gene as identified by WGS. This prevalence study shows that mcr-1 is present in low levels patients out of the community attending a hospital. Furthermore the study underlines the importance of phenotypical confirmation of molecular detection of a mcr-1 gene.

Detection of Clostridium difficile in feces of asymptomatic patients admitted to the hospital

ABSTRACT Recent evidence shows that patients asymptomatically colonized with Clostridium difficile may contribute to the transmission of C. difficile in health care facilities. Additionally, these patients may have a higher risk of developing C. difficile infection. The aim of this study was to compare a commercially available PCR directed to both toxin A and B (artusC. difficile QS-RGQ kit CE; Qiagen), an enzyme-linked fluorescent assay to glutamate dehydrogenase (GDH ELFA) (Vidas, bioMérieux), and an in-house-developed PCR to tcdB, with (toxigenic) culture of C. difficile as the gold standard to detect asymptomatic colonization. Test performances were evaluated in a collection of 765 stool samples obtained from asymptomatic patients at admission to the hospital. The C. difficile prevalence in this collection was 5.1%, and 3.1% contained toxigenic C. difficile. Compared to C. difficile culture, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the C. difficile GDH ELFA were 87.2%, 91.2%, 34.7%, and 99.3%, respectively. Compared with results of toxigenic culture, the sensitivity, specificity, PPV, and NPV of the commercially available PCR and the in-house PCR were 95.8%, 93.4%, 31.9%, 99.9%, and 87.5%, 98.8%, 70%, and 99.6%, respectively. We conclude that in a low-prevalence setting of asymptomatically colonized patients, both GDH ELFA and a nucleic acid amplification test can be applied as a first screening test, as they both display a high NPV. However, the low PPV of the tests hinders the use of these assays as stand-alone tests.

Fecal Microbiota Transfer for Multidrug-Resistant Gram-Negatives: A Clinical Success Combined With Microbiological Failure.

Abstract Combined fecal microbiota transfer and antibiotic treatment prevented recurrences of urinary tract infections with multidrug-resistant (MDR) Pseudomonas aeruginosa, but it failed to eradicate intestinal colonization with MDR Escherichia coli. Based on microbiota analysis, failure was not associated with distinct diminished microbiota diversity.

Effect of Detecting and Isolating Asymptomatic Clostridium difficile Carriers

“Don’trecommendcancerscreeninginadultswithlifeexpectancy of less than 10 years” and find clear statistics and graphics in a decision aid. The Mayo clinic has created such a resource that could serve as a model for shared decision-making tools.10 In addition to providing tools and training, the ABIM Foundation should incorporate best practices in communication into testing for accreditationandmaintenanceofcertification.BybuildingChoosingWiselyintoastreamlinedresourcethattiesrecommendations to evidence and using accreditation programs to reinforce communication skills, the ABIM Foundation would enable an informed conversation that balances risks and benefits and might deter overuse. Thanks in part to campaigns like JAMA Internal Medicine’s Less Is More and Choosing Wisely, high-value care has been adopted as a professional responsibility. The story of how we implement this in practice is yet to unfold. More attention to numeracy and shared decision-making communication skills will complement system interventions like deployment of effective EMR-based decision support tools at the point of care. Physicians at all levels of training must take on the additional professional obligation of communicating risk and benefits clearly to achieve care that is both high-value and in line with patients’ goals.

Faecal microbiota transplantation in clinical practice

Dear Sir, We would like to add some remarks to the report of a consensus meeting about faecal microbiota transplantation (FMT) by Cammarota et al. 1 Already, donor faeces banks exist at an institutional or national level in Germany, UK and The Netherlands, to support treatment of patients with recurrent Clostridium difficile infection (CDI).2 Unfortunately, these centres were not consulted for advice, and it is felt that some conclusions of the report need clarification and adjustments. First, the statement about expert centres is inaccurate. The critical steps for safe and effective FMT are (1) patient selection, (2) donor (stool) selection and screening, and (3) biobanking of faeces suspensions. We agree that donor screening should be performed …

Is the Lower Gastrointestinal Route Really Preferred Over the Upper Gastrointestinal Route for Fecal Microbiota Transfer

To the Editor: Furuya-Kanamori et al1 recently published a paper entitled “Upper Versus Lower Gastrointestinal Delivery for Transplantation of Fecal Microbiota in Recurrent or Refractory Clostridium difficile Infection: A Collaborative Analysis of Individual Patient Data From 14 Studies” in the Journal of Clinical Gastroenterology. We question if the authors’ conclusion that the lower gastrointestinal (LGI) route is preferred over the upper gastrointestinal (UGI) route in treatment of recurrent Clostridium difficile infection, is a correct interpretation of the literature analyzed. First, the patient groups differ in baseline characteristics. Substantially more children have been included in the UGI group (12%) than in the LGI group (3%). Fecal microbiota transfer (FMT) has not been well studied in the pediatric population, and its efficacy needs to be established before pooling data with adults. No adjustment has been made for possible confounders other than age and sex (such as comorbidity, antibiotic use after FMT, and number of FMT treatments), which can influence the relapse rate significantly. Second, the article does not describe a thorough quality assessment of the studies included in the metaanalysis. For instance, the authors did not address the fact that more than half of the patients received less than the recommended 50 g of donor feces, as Gough et al,2 demonstrated a 4 times greater relapse rate when <50g of stool was used. In contrast, data of the first published randomized controlled trial of FMT by van Nood et al,3 is not included in this meta-analysis. Third, the treatment effect of the LGI and UGI route only diverges from 30 days onwards, and not directly after 30 days as suggested in the article. The median follow-up period was 80 days in the UGI and 354 days in the LGItreated patients. Therefore, the difference between the groups at 90 days follow-up is based on a very small sample of patients. As there is no biological plausibility for the superiority of the LGI route, and the temporality of the observed effect opposes a direct treatment effect, we suppose that this outcome may have been a result of bias. Since 2008, we have treated several dozens of patients in the Netherlands with FMT via a nasoduodenal tube. We have not noticed any recurrences 30 days after FMT, with a median follow-up time of 21 months (van Beurden et al, manuscript submitted). We prefer the UGI route because it is generally well tolerated by patients, and less invasive compared with colonoscopy, especially in an inflamed bowel as with severe Clostridium difficile infection. We agree with the authors that a randomized controlled trial with a sufficient sample size is required to determine the optimal route for administration of FMT.

Mechanistic Insights in the Success of Fecal Microbiota Transplants for the Treatment of Clostridium difficile Infections

Fecal microbiota transplantation has proven to be an effective treatment for infections with the gram-positive enteropathogen Clostridium difficile. Despite its effectiveness, the exact mechanisms that underlie its success are largely unclear. In this review, we highlight the pleiotropic effectors that are transferred during fecal microbiota transfer and relate this to the C. difficile lifecycle. In doing so, we show that it is likely that multiple factors contribute to the elimination of symptoms of C. difficile infections after fecal microbiota transplantation.