Clostridium difficile infection: Early history, diagnosis and molecular strain typing methods.

Recognised as the leading cause of nosocomial antibiotic-associated diarrhoea, the incidence of Clostridium difficile infection (CDI) remains high despite efforts to improve prevention and reduce the spread of the bacterium in healthcare settings. In the last decade, many studies have focused on the epidemiology and rapid diagnosis of CDI. In addition, different typing methods have been developed for epidemiological studies. This review explores the history of C. difficile and the current scope of the infection. The variety of available laboratory tests for CDI diagnosis and strain typing methods are also examined.

Clostridium difficile presence in Spanish and Belgian hospitals.

Clostridium difficile is recognised worldwide as the main cause of infectious bacterial antibiotic-associated diarrhoea in hospitals and other healthcare settings. The aim of this study was to first survey C. difficile prevalence during the summer of 2014 at the Central University Hospital of Asturias (Spain). By typing the isolates obtained, it was then possible to compare the ribotype distribution at the Spanish hospital with results from the St Luc University Hospital in Belgium over the same period. The prevalence of positive cases reported in Spain and Belgium was 12.3% and 9.3% respectively. The main PCR-ribotypes previously described in Europe were found in both hospitals, including 078, 014, 012, 020 and 002. In the Spanish hospital, most of the C. difficile-positive samples were referred from oncology, acute care and general medicine services. In the Belgian hospital the majority of positive samples were referred from the paediatric service. However, a high percentage of isolates from this service were non-toxigenic. This study finds that the presence and detection of C. difficile in paediatric and oncology services requires further investigation.

Clostridium difficile in Food and Animals: A Comprehensive Review.

Zoonoses are infections or diseases that can be transmitted between animals and humans through direct contact, close proximity or the environment. Clostridium difficile is ubiquitous in the environment, and the bacterium is able to colonise the intestinal tract of both animals and humans. Since domestic and food animals frequently test positive for toxigenic C. difficile, even without showing any signs of disease, it seems plausible that C. difficile could be zoonotic. Therefore, animals could play an essential role as carriers of the bacterium. In addition, the presence of the spores in different meats, fish, fruits and vegetables suggests a risk of foodborne transmission. This review summarises the current available data on C. difficile in animals and foods, from when the bacterium was first described up to the present.

Clostridium difficile infection and intestinal microbiota interactions.

Clostridium difficile remains the leading cause of healthcare-associated diarrhoea and outbreaks continue to occur worldwide. Aside from nosocomial C. difficile infection, the bacterium is also increasingly important as a community pathogen. Furthermore, asymptomatic carriage of C. difficile in neonates, adults and animals is also well recognised. The investigation of the gut's microbial communities, in both healthy subjects and patients suffering C. difficile infection (CDI), provides findings and information relevant for developing new successful approaches for its treatment, such as faecal microbiota transplantation, or for the prophylaxis of the infection by modification of the gut microbiota using functional foods and beverages. The analysis of all available data shows new insights into the role of intestinal microbiota in health and disease.

Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture.

Speeding up the turn-around time of positive blood culture identifications is essential in order to optimize the treatment of septic patients. Several sample preparation techniques have been developed allowing direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of positive blood cultures. Yet, the hands-on time restrains their routine workflow. In this study, we evaluated an approach whereby MALDI-TOF MS identification without any additional steps was carried out on short subcultured colonies from positive blood bottles with the objective of allowing results reporting on the day of positivity detection. Over a 7-month period in 2012, positive blood cultures detected by 9 am with an automated system were inoculated onto a Columbia blood agar and processed after a 5-h incubation on a MALDI-TOF MicroFlex platform (Bruker Daltonik GmbH). Single-spotted colonies were covered with 1 µl formic acid and 1 µl matrix solution. The results were compared to the validated identification techniques. A total of 925 positive blood culture bottles (representing 470 bacteremic episodes) were included. Concordant identification was obtained in 727 (81.1 %) of the 896 monomicrobial blood cultures, with failure being mostly observed with anaerobes and yeasts. In 17 episodes of polymicrobic bacteremia, the identification of one of the two isolates was achieved in 24/29 (82.7 %) positive cultures. Routine implementation of MALDI-TOF MS identification on young positive blood subcultures provides correct results to the clinician in more than 80 % of the bacteremic episodes and allows access to identification results on the day of blood culture positivity detection, potentially accelerating the implementation of targeted clinical treatments.

Clostridium difficile from food and surface samples in a Belgian nursing home: an unlikely source of contamination.

This study investigates the contamination of foods and surfaces with Clostridium difficile in a single nursing home. C. difficile PCR-ribotype 078 was found in one food sample and in none of the tested surfaces. These results indicate that food and surfaces are an unlikely source of C. difficile infection in this setting.

Multilocus sequence typing analysis and antibiotic resistance of Clostridium difficile strains isolated from retail meat and humans in Belgium.

Clostridium difficile has been isolated from food animals and meat, specially ground pork and ground beef. The recovered isolates were closely related to C. difficile human strains, indicating that animals and food are possible transmission routes of human C. difficile infection. The main objective of this study was to characterize C. difficile isolates from retail meat and to compare with human isolates recovered from hospital patients in Belgium. Raw meat (beef and pork) was obtained from the retail trade. C. difficile was recovered from 2.3% of the beef samples and from 4.7% of the pork samples. A total of 4 different PCR-ribotypes were identified with a large percentage of types 078 and 014. Resistance to moxifloxacin and erythromycin was detected. The multi-locus sequence typing (MLST) analysis showed that meat and human isolates cluster in the same lineage. This study reveals the presence of toxigenic C. difficile in retail meat in Belgium with predominance PCR-ribotypes 078 and 014, which are among the four most prevalent ribotypes of C. difficile isolated from humans in Europe.

Clostridium difficile infection in elderly nursing home residents.

Age-related changes in intestinal flora and host defences, the receipt of antibiotic treatment, and the presence of underlying diseases are some of the most common risk factors associated with Clostridium difficile infection. Therefore, retirement care facilities for elderly people have been pinpointed as frequent sources of contamination. There is only limited data regarding the presence and epidemiology of C. difficile in nursing homes, and this gap in the current literature emphasises the need to gain a better understanding of the situation in order to prevent the emergence of new outbreaks among this population group.

Impact of ribotype 027 on Clostridium difficile infection in a geriatric department.

The purposes of this study were to describe the epidemiology (2001-2009) of Clostridium difficile infections (CDI) in a geriatric department and to compare the clinical data of patients infected with a 027 or non-027 strain. We retrospectively identified all geriatric patients with CDI and analysed the clinical and microbiological data of 133 patients for whom a ribotype was available between March 2003 and December 2009. The incidence of CDI in our geriatric department increased from 0.2 per 100 admissions in 2001 to 8.1 in 2004 and decreased to 1.3 in 2008 before a new rise to 2.1 in 2009. The percentage of ribotype 027 decreased from 2007 but it remained the most prevalent ribotype during the years 2007-2009, with a greater dispersion of ribotypes. The mean age of the patients was 84 years and the median Charlson index was 6.0. Previous use of fluoroquinolones was a significant risk factor for developing a CDI with an 027 strain (p = 0.001). Cure was significantly lower in the 027 group (p = 0.003). The total attributable mortality was 24.1 %. A multiparametric model showed that attributable mortality was influenced by the ribotype 027 (p = 0.037), the severity of clinical symptoms (p = 0.001) and the type of treatment (p = 0.002). Oral vancomycin had a protective effect against mortality. Attention should be paid to elderly patients developing a CDI, especially after the administration of fluoroquinolones. Oral vancomycin could be recommended as the first-line agent not only to protect against recurrence or severe CDI, but to diminish the attributable mortality risk.

Clostridium difficile in young farm animals and slaughter animals in Belgium.

Faecal carriage of Clostridium difficile in healthy animals has been reported recently, especially in piglets and calves. However there is limited data about carriage in animals just prior to slaughter in Europe. The main objective of this study was to determine the presence of C. difficile in pigs and cattle at the slaughterhouse. C. difficile was isolated in 6.9% of the cattle at the slaughterhouse. None of the pig slaughter samples were positive for C. difficile after an enrichment time of 72 h. For complementary data, a short study was conducted in piglets and calves at farms. C. difficile was more prevalent in piglets (78.3%) than in calves (22.2%) on the farms. Regarding the piglet samples, 27.8% of the positive samples were detected without enrichment of stools. The PCR ribotype 078 was predominant in farm animals. Samples isolated from slaughter cattle presented the widest range in PCR-ribotype variety, and the most prevalent PCR ribotype was 118a UCL. The results of this study confirm that C. difficile is present in slaughter animals in Belgium with a large percentage of toxigenic strains also commonly found in humans.

Comparison of a commercially available repetitive-element PCR system (DiversiLab) with PCR ribotyping for typing of clostridium difficile strains.

This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

Nosocomial and non-nosocomial Clostridium difficile infections in hospitalised patients in Belgium: compulsory surveillance data from 2008 to 2010.

Surveillance of Clostridium difficile infection (CDI) is compulsory in Belgian hospitals. Our objectives were to compare incidence and case characteristics of nosocomial infections (Nc-CDI) with onset of diarrhoea more than two days after hospital admission, with non-nosocomial cases (Nnc-CDI). The database included inpatients from 2008 to 2010. Of 8,351 cases reported by 150 hospitals, 3,102 (37%) were classified as Nnc-CDI and 5,249 (63%) as Nc-CDI. In 2010, the mean incidence per 1,000 admissions was 0.95 for Nc-CDI and 0.56 for Nnc-CDI. Both incidences were relatively stable over the three years, with a slight decrease in 2010 (p<0.01). Onset of symptoms in Nnc- CDI cases took place in the community (57.1%), nursing homes (14.2%) or hospitals (17.5%); data for 11.2%were missing. Nnc-CDI cases were younger than Nc-CDI (median age 75 vs. 79 years, p<0.001), and more likely to be women (62% vs. 57%, p<0.001) and to have pseudomembranous colitis (5.3% vs. 1.6%, p<0.001). In 2009, C. difficile ribotype 027 was found in 32 of 70 reporting hospitals compared with 19 of 69 in 2010 (p<0.03). Although our study population only included hospitalised patients, the results do not support the hypothesis of an increase in the incidence of severe community-associated CDI.

Nation-wide prospective surveillance of Clostridium difficile infections in hospitals in Belgium, July 2007-June 2008.

We report here baseline data from the first year of compulsory surveillance of Clostridium difficile infections (CDI) in hospitals in Belgium. Between 1 July 2007 and 30 June 2008, 2,704 CDI were reported: 12% were recurrent and 66% were hospital-associated (half of which occurred 15 days or more after admission). CDI was considered the cause of death (direct or indirect) for 10% of the episodes. The median incidence of CDI was 1.5 per 1,000 admissions and 1.9 per 10,000 hospital-days for all cases, and 0.9 per 1,000 admissions, and 1.1 per 10,000 hospital-days for hospital-associated cases. Further investigation of risk stratification by average length of stay in the reporting hospitals is warranted as a way to improve the comparability of indicators across hospitals and surveillance systems. In spite of methodological issues, the surveillance of CDI in Belgian hospitals has been able to produce robust baseline data that should allow monitoring of trends at hospital and national level, and provide a basis for international comparisons. Remaining challenges are to define and monitor targets for the control of CDI, and to improve the individual feed-back of data at hospital level.

Clostridium difficile beyond stools: dog nasal discharge as a possible new vector of bacterial transmission.

Zoonotic transmission of Clostridium difficile has been largely hypothesised to occur after direct or indirect contact with contaminated animal faeces. Recent studies have reported the presence of the bacterium in the natural environment, including in soils and rivers. If C. difficile spores are scattered in the environment, they can easily enter the respiratory tract of dogs, and therefore, dog nasal discharge could be a direct route of transmission not previously investigated. This study reports for the first time the presence of C. difficile in the respiratory tracts of dogs. The bacterium was isolated from 6 (17.1%) out of 35 nasal samples, with a total of 4 positive dogs (19%). C. difficile was recovered from both proximal and distal nasal cavities. All isolates were toxigenic and belonged to PCR-ribotype 014, which is one of the most predominant types in animals and in community-acquired C. difficile infections in recent years. The findings of this study demonstrate that the nasal cavity of dogs is contaminated with toxigenic C. difficile, and therefore, its secretions could be considered as a new route by which bacteria are spread and transmitted.

Corrigendum to "Clostridium difficile beyond stools: dog nasal discharge as a possible new vector of bacterial transmission" [Heliyon 5 (5) (May 2019) e01629].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e01629.].

Update of Clostridium difficile infection due to PCR ribotype 027 in Europe, 2008.

Outbreaks of Clostridium difficile infections (CDI) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America and Europe. This emerging strain is referred to as PCR ribotype 027 (Type 027). Since 2005, individual countries have developed surveillance studies about the spread of type 027.C. difficile Type 027 has been reported in 16 European countries. It has been responsible for outbreaks in Belgium, Germany, Finland, France, Ireland, Luxembourg, The Netherlands, Switzerland and the United Kingdom (England, Wales, Northern Ireland and Scotland). It has also been detected in Austria, Denmark, Sweden, Norway, Hungary, Poland and Spain. Three countries experienced imported patients with CDI due to Type 027 who acquired the infection abroad.The antimicrobial resistance pattern is changing, and outbreaks due to clindamycin-resistant ermB positive Type 027 strains have occurred in three European countries. Ongoing epidemiological surveillance of cases of CDI, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of new, highly virulent clones.

Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates.

A 2-month prospective study of Clostridium difficile infections was conducted in 38 hospitals from 14 different European countries in order to obtain an overview of the phenotypic and genotypic features of clinical isolates of C. difficile during 2005. Of 411 isolates from diarrhoeagenic patients with suspected C. difficile-associated diarrhoea (CDAD), 354 were toxigenic, of which 86 (24.3%) were toxin-variant strains. Major toxinotypes included toxinotypes 0 (n = 268), V (n = 28), VIII (n = 22) and III (n = 25). MICs of metronidazole, vancomycin, erythromycin, clindamycin, moxifloxacin and tetracycline were determined using the Etest method. All the toxigenic strains were fully-susceptible to metronidazole and vancomycin. Resistance to erythromycin, clindamycin, tetracycline and moxifloxacin was found in 44.4%, 46.1%, 9.2% and 37.5% of the isolates, respectively. Sixty-six different PCR ribotypes were characterised, with the 027 epidemic strain accounting for 6.2% of isolates. This strain was positive for binary toxin genes, had an 18-bp deletion in the tcdC gene, and was resistant to both erythromycin and moxifloxacin. The mean incidence of CDAD was 2.45 cases/10 000 patient-days, but this figure varied widely among the participating hospitals. Patients infected with the 027 strain were more likely to have a severe disease (OR 3.29, 95% CI 1.19-9.16, p 0.008) and to have been specifically treated with metronidazole or vancomycin (OR 7.46, 95% CI 1.02-154, p 0.02). Ongoing epidemiological surveillance of cases of CDAD, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of specific highly virulent clones.

Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe.

Recent outbreaks of Clostridium difficile-associated diarrhoea (CDAD) with increased severity, high relapse rate and significant mortality have been related to the emergence of a new, hypervirulent C. difficile strain in North America, Japan and Europe. Definitions have been proposed by the European Centre of Disease Prevention and Control (ECDC) to identify severe cases of CDAD and to differentiate community-acquired cases from nosocomial CDAD (http://www.ecdc.europa.eu/documents/pdf/Cl_dif_v2.pdf). CDAD is mainly known as a healthcare-associated disease, but it is also increasingly recognised as a community-associated disease. The emerging strain is referred to as North American pulsed-field type 1 (NAP1) and PCR ribotype 027. Since 2005, individual countries have developed surveillance studies to monitor the spread of this strain. C. difficile type 027 has caused outbreaks in England and Wales, Ireland, the Netherlands, Belgium, Luxembourg, and France, and has also been detected in Austria, Scotland, Switzerland, Poland and Denmark. Preliminary data indicated that type 027 was already present in historical isolates collected in Sweden between 1997 and 2001.

Clinical and laboratory features of the most common Clostridium difficile ribotypes isolated in Belgium.

BACKGROUND: Previous studies comparing Clostridium difficile infection (CDI) due to different ribotypes have been conflicting, and many have only compared small numbers of cases or few ribotypes. AIM: To compare patient and episode characteristics for CDI due to different ribotypes. METHODS: The ribotyping results from 3333 toxin-producing isolates collected from 110 Belgian hospitals between October 2010 and December 2015 were matched to clinical data from the national CDI surveillance database. Data for ribotypes with at least 100 occurrences were compared. In addition, the national reference laboratory quantitatively measured the level of toxin production in five randomly chosen cultured isolates for each of the most common ribotypes. FINDINGS: Ribotypes with more than 100 occurrences were R014, R020, R002, R078, R027, R005 and R106 (Brazier classification). The median age for all patients was 79 years [patients with R027, 83 years (P<0.001); patients with R106, 73 years (P<0.001)]. In total, 10% of episodes were recurrences; values were higher for R027 (22%) and R106 (18%). CDI due to R078 was not significantly more likely to be community associated than healthcare associated (28% vs 24%; P=0.1). Complications occurred in 7% of all episodes, and 12% for those with R027 and R078. However, after adjusting for age, onset outside the hospital and recurrence, R027 was no longer associated with complications [odds ratio (OR) 1.3, 95% confidence interval (CI) 0.7-2.4], unlike R078 (OR 1.7, 95% CI 1.0-2.6; P=0.04). A positive stool toxin test and greater levels of toxin production in the cultured isolates were more likely for R078 and R027. CONCLUSION: Out of the seven most common ribotypes in hospital patients, R078 and R027 were associated with higher rates of complications. Infections with R027 and R106 were more likely to be recurrent. The presence of toxin in stools was most likely with R078, R027 and R106, with highest levels of toxin production in vitro for R078 and R027. R060 produced the lowest levels of toxin in vitro.

Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays.

OBJECTIVES: Neither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. METHODS: We designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. RESULTS: The technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. CONCLUSIONS: We propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories.