Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridioides difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC), should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays or single-molecule array assays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has been advocated by international guidelines (IDSA/SHEA and ESCMID) in order to optimize diagnostic accuracy. As a result, a survey performed in 2018-2019 in Europe revealed that most of all hospital sites reported using more than one test to diagnose CDI. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal microbiota biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.

Understanding Clostridium difficile Colonization.

Clostridium difficile is the main causative agent of antibiotic-associated and health care-associated infective diarrhea. Recently, there has been growing interest in alternative sources of C. difficile other than patients with Clostridium difficile infection (CDI) and the hospital environment. Notably, the role of C. difficile-colonized patients as a possible source of transmission has received attention. In this review, we present a comprehensive overview of the current understanding of C. difficile colonization. Findings from gut microbiota studies yield more insights into determinants that are important for acquiring or resisting colonization and progression to CDI. In discussions on the prevalence of C. difficile colonization among populations and its associated risk factors, colonized patients at hospital admission merit more attention, as findings from the literature have pointed to their role in both health care-associated transmission of C. difficile and a higher risk of progression to CDI once admitted. C. difficile colonization among patients at admission may have clinical implications, although further research is needed to identify if interventions are beneficial for preventing transmission or overcoming progression to CDI.

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridium difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC) should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has now been advocated by international guidelines in order to optimize diagnostic accuracy. Despite these recommendations, testing methods between hospitals vary widely, which impacts CDI incidence rates. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Detection of Clostridium difficile in Feces of Asymptomatic Patients Admitted to the Hospital.

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 (artus C. 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.

Fungal and bacterial gut microbiota differ between Clostridioides difficile colonization and infection.

Aim: The bacterial microbiota is well-recognized for its role in Clostridioides difficile colonization and infection, while fungi and yeasts remain understudied. The aim of this study was to analyze the predictive value of the mycobiota and its interactions with the bacterial microbiota in light of C. difficile colonization and infection. Methods: The mycobiota was profiled by ITS2 sequencing of fecal DNA from C. difficile infection (CDI) patients (n = 29), asymptomatically C. difficile colonization (CDC) patients (n = 38), and hospitalized controls with C. difficile negative stool culture (controls; n = 38). Previously published 16S rRNA gene sequencing data of the same cohort were used additionally for machine learning and fungal-bacterial network analysis. Results: CDI patients were characterized by a significantly higher abundance of Candida spp. (MD 0.270 ± 0.089, P = 0.002) and Candida albicans (MD 0.165 ± 0.082, P = 0.023) compared to controls. Additionally, they were deprived of Aspergillus spp. (MD -0.067 ± 0.026, P = 0.000) and Penicillium spp. (MD -0.118 ± 0.043, P = 0.000) compared to CDC patients. Network analysis revealed a positive association between several fungi and bacteria in CDI and CDC, although the analysis did not reveal a direct association between Clostridioides spp. and fungi. Furthermore, the microbiota machine learning model outperformed the models based on the mycobiota and the joint microbiota-mycobiota model. The microbiota classifier successfully distinguished CDI from CDC [Area Under the Receiver Operating Characteristic (AUROC) = 0.884] and CDI from controls (AUROC = 0.905). Blautia and Bifidobacterium were marker genera associated with CDC patients and controls. Conclusion: The gut mycobiota differs between CDI, CDC, and controls and may affect Clostridioides spp. through indirect interactions. The mycobiota data alone could not successfully discriminate CDC from controls or CDI patients and did not have additional predictive value to the bacterial microbiota data. The identification of bacterial marker genera associated with CDC and controls warrants further investigation.

Hestia criteria can discriminate high- from low-risk patients with pulmonary embolism.

We investigated whether the clinical criteria used in the Hestia study for selection of pulmonary embolism (PE) patients for outpatient treatment could discriminate PE patients with high and low risk for adverse clinical outcome. We performed a cohort study with PE patients who were triaged with 11 criteria for outpatient treatment. Patients not eligible for outpatient treatment were treated in hospital. Study outcomes were recurrent venous thromboembolism, major bleeding and all-cause mortality during 3 months. In total, 530 patients were included, of which 297 were treated at home. In the outpatient group, six patients (2.0%, 95% CI 0.7-4.3%) had recurrent venous thromboembolism versus nine in-patients (3.9%, 95% CI 1.9-7.0%). Three patients (1.0%, 95% CI 0.2-2.9) died during the 3-months follow-up in the outpatient group versus 22 patients (9.6%, 95% CI 6.3-14) in the in-patient group (p<0.05). None of the outpatients died as a result of fatal PE versus five (2.2%) in-patients (p<0.05). In the outpatient group, 0.7% (95% CI 0.08-2.4) had major bleeding events versus 4.8% (95% CI 2.4-8.4) of in-patients (p<0.05). This study showed that the Hestia criteria can discriminate PE patients with low risk from patients with high risk for adverse clinical outcome. The low-risk patients can safely be treated at home.

Screening for Clostridioides difficile colonization at admission to the hospital: a multi-centre study.

OBJECTIVES: To assess the value of screening for Clostridioides difficile colonization (CDC) at hospital admission in an endemic setting. METHODS: A multi-centre study was conducted at four hospitals located across the Netherlands. Newly admitted patients were screened for CDC. The risk of development of Clostridioides difficile infection (CDI) during admission and 1-year follow-up was assessed in patients with and without colonization. C. difficile isolates from patients with colonization were compared with isolates from incident CDI cases using core genome multi-locus sequence typing to determine whether onwards transmission had occurred. RESULTS: CDC was present in 108 of 2211 admissions (4.9%), whereas colonization with a toxigenic strain (toxigenic Clostridoides difficile colonization [tCDC]) was present in 68 of 2211 admissions (3.1%). Among these 108 patients with colonization, diverse PCR ribotypes were found and no 'hypervirulent' PCR ribotype 027 (RT027) was detected (95% CI, 0-0.028). None of the patients with colonization developed CDI during admission (0/49; 95% CI, 0-0.073) or 1-year follow-up (0/38; 95% CI, 0-0.93). Core genome multi-locus sequence typing identified six clusters with genetically related isolates from patients with tCDC and CDI; however, in these clusters, only one possible transmission event from a patient with tCDC to a patient with CDI was identified based on epidemiological data. CONCLUSION: In this endemic setting with a low prevalence of 'hypervirulent' strains, screening for CDC at admission did not detect any patients with CDC who progressed to symptomatic CDI and detected only one possible transmission event from a patient with colonization to a patient with CDI. Thus, screening for CDC at admission is not useful in this setting.

Survival after cancer-related venous thrombosis: the Scandinavian Thrombosis and Cancer Study.

Patients with cancer have an increased risk of developing venous thromboembolism (VTE), and this combination is reported to result in poorer survival compared with cancer alone. This study aimed to investigate the impact of VTE on the survival of patients with cancer in a general population. The Scandinavian Thrombosis and Cancer (STAC) cohort, a population-based cohort including 144 952 participants without previous VTE or cancer, was used. During follow-up, cancer and VTE incidences were registered. "Cancer-related VTE" was defined as VTE diagnosed in patients with overt or occult cancer. The survival of participants without cancer and/or VTE ("disease-free") was compared with the survival of participants with cancer and cancer-related VTE. Cox regression models with cancer and VTE as time-varying exposures were performed to calculate hazard ratios for death. Subanalyses were performed across cancer types and stages and VTE type (deep vein thrombosis or pulmonary embolism). During follow-up (mean, 11.7 years), 14 621 participants developed cancer, and 2444 developed VTE, of which 1241 were cancer-related. The mortality rates (per 100 person years) for disease-free participants, VTE only, cancer only, and cancer-related VTE were 0.63, 5.0, 9.2, and 45.3, respectively. Compared with patients with cancer only, the risk of death for patients with cancer-related VTE was increased 3.4-fold. Within all cancer types, the occurrence of VTE increased the mortality risk 2.8- to 14.7-fold. In a general population, patients with cancer with VTE had a 3.4-fold higher mortality risk than patients with cancer without VTE, independent of cancer type.

The Bacterial Gut Microbiota of Adult Patients Infected, Colonized or Noncolonized by Clostridioides difficile.

: Gut microbiota composition in patients with Clostridioides difficile colonization is not well investigated. We aimed to identify bacterial signatures associated with resistance and susceptibility to C. difficile colonization (CDC) and infection (CDI). Therefore, gut microbiota composition from patients with CDC (n = 41), with CDI (n = 41), and without CDC (controls, n = 43) was determined through 16S rRNA gene amplicon sequencing. Bacterial diversity was decreased in CDC and CDI patients (p<0.01). Overall microbiota composition was significantly different between control, CDC, and CDI patients (p = 0.001). Relative abundance of Clostridioides (most likely C. difficile) increased stepwise from controls to CDC and CDI patients. In addition, differential abundance analysis revealed that CDI patients' gut microbiota was characterized by significantly higher relative abundance of Bacteroides and Veillonella than CDC patients and controls. Control patients had significantly higher Eubacterium hallii and Fusicatenibacter abundance than colonized patients. Network analysis indicated that Fusicatenibacter was negatively associated with Clostridioides in CDI patients, while Veillonella was positively associated with Clostridioides in CDC patients. Bacterial microbiota diversity decreased in both CDC and CDI patients, but harbored a distinct microbiota. Eubacterium hallii and Fusicatenibacter may indicate resistance against C. difficile colonization and subsequent infection, while Veillonella may indicate susceptibility to colonization and infection by C. difficile.

Application of Antibody-Mediated Therapy for Treatment and Prevention of Clostridium difficile Infection.

Clostridium difficile causes antibiotic- and healthcare-associated diarrhea, which is characterized by a high mortality rate (5-15%) and high recurrence rate of 20% or more. Therapeutic alternatives to antibiotics are urgently needed to improve the overall cure rate. Among these, therapeutic antibodies have shown promising results in clinical studies. Herein, the authors review current monoclonal and polyclonal anti- C. difficile antibodies that have entered the clinical development stage, either for systemic administration or by the oral route. The antibodies can be applied as monotherapy or in combination with standard-of-care to treat an infection with C. difficile or to protect from a recurrence. Bezlotoxumab is the first antibody for secondary prevention of recurrence of C. difficile infection approved by the regulatory agencies in US and Europe. The human monoclonal antibody is administered systemically to patients receiving oral standard-of-care antibiotics. Other antibodies are currently in the clinical pipeline, and some are intended for oral use. They show a good safety profile, high efficacy and low production costs, and can be considered promising therapies of the future. The most promising orally administered drug candidate is a bovine antibody from hyperimmune colostral milk, which is in an advanced clinical development stage. Which antibody will enter the market is dependent on its bioavailability at the site of infection as well as its activity against C. difficile toxins, protection against colonization and possible action on spore formation. The antibody must demonstrate a clear benefit in comparison with other available treatment options to be considered for use by clinicians.

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.

[Thromboprophylaxis in outpatients with cancer?]. / Tromboseprofylaxe bij ambulante patiënten met kanker?

Outpatients with cancer have an increased risk of venous thrombosis, especially during chemotherapy. Clinical trials have shown that thromboprophylaxis with low-molecular-weight heparin reduces the incidence of symptomatic venous thrombosis in these patients. An increase in major bleeding events was suggested but not confirmed in most recent trials. However, as the incidence of venous thrombosis is relatively low in the general cancer population, thromboprophylaxis should not be recommended for all cancer outpatients. Instead, to optimize the risk/benefit ratio, targeted thromboprophylaxis for patients deemed at high risk is recommended. Risk prediction models may be used to identify these high risk patients. Until results of clinical trials based on these models are published, thromboprophylaxis can be considered in patients with advanced or metastatic high risk cancers (e.g. pancreatic and lung cancer) who are receiving chemotherapy and have no increased risk of bleeding. Because of the many uncertainties, the decision to start thromboprophylaxis should always be discussed with the patient.

Comparison of two methods for selection of out of hospital treatment in patients with acute pulmonary embolism.

The aim of this study is to compare the performance of two clinical decision rules to select patients with acute pulmonary embolism (PE) for outpatient treatment: the Hestia criteria and the simplified Pulmonary Embolism Severity Index (sPESI). From 2008 to 2010, 468 patients with PE were triaged with the Hestia criteria for outpatient treatment: 247 PE patients were treated at home and 221 were treated as inpatients. The outcome of interest was all-cause 30-day mortality. In a post-hoc fashion, the sPESI items were scored and patients were classified according to the sPESI in low and high risk groups. Of the 247 patients treated at home, 189 (77%) patients were classified as low risk according to the sPESI and 58 patients (23%) as high risk. In total, 11 patients died during the first month; two patients treated at home and nine patients treated in-hospital. None of the patients treated at home died of fatal PE. Both the Hestia criteria and sPESI selected >50% of patients as low risk, with good sensitivity and negative predictive values for 30-day mortality: 82% and 99% for the Hestia criteria and 91% and 100% for the sPESI, respectively. The Hestia criteria and the sPESI classified different patients eligible for outpatient treatment, with similar low risks for 30-day mortality. This study suggests that the Hestia criteria may identify a proportion of high risk sPESI patiennts who can be safely treated at home, this however requires further validation.