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.].

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.

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.

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.

Connectivity of diagnostic technologies: improving surveillance and accelerating tuberculosis elimination.

In regard to tuberculosis (TB) and other major global epidemics, the use of new diagnostic tests is increasing dramatically, including in resource-limited countries. Although there has never been as much digital information generated, this data source has not been exploited to its full potential. In this opinion paper, we discuss lessons learned from the global scale-up of these laboratory devices and the pathway to tapping the potential of laboratory-generated information in the field of TB by using connectivity. Responding to the demand for connectivity, innovative third-party players have proposed solutions that have been widely adopted by field users of the Xpert(®) MTB/RIF assay. The experience associated with the utilisation of these systems, which facilitate the monitoring of wide laboratory networks, stressed the need for a more global and comprehensive approach to diagnostic connectivity. In addition to facilitating the reporting of test results, the mobility of digital information allows the sharing of information generated in programme settings. When they become easily accessible, these data can be used to improve patient care, disease surveillance and drug discovery. They should therefore be considered as a public health good. We list several examples of concrete initiatives that should allow data sources to be combined to improve the understanding of the epidemic, support the operational response and, finally, accelerate TB elimination. With the many opportunities that the pooling of data associated with the TB epidemic can provide, pooling of this information at an international level has become an absolute priority.

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: 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 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.

Investigation of Clostridium difficile interspecies relatedness using multilocus sequence typing, multilocus variable-number tandem-repeat analysis and antimicrobial susceptibility testing.

Multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA) and antimicrobial susceptibility were performed on 37 animal and human C. difficile isolates belonging to 15 different PCR-ribotypes in order to investigate the relatedness of human and animal isolates and to identify possible transmission routes. MLVA identified a total of 21 different types while MLST only distinguished 12 types. Identical C. difficile strains were detected in the same animal species for PCR-ribotypes 014, 078, UCL 16U and UCL 36, irrespective of their origin or the isolation date. Non clonal strains were found among different hosts; however, a high genetic association between pig and cattle isolates belonging to PCR-ribotype 078 was revealed. MLVA also showed genetic differences that clearly distinguished human from animal strains. For a given PCR-ribotype, human and animal strains presented a similar susceptibility to the antimicrobials tested. All strains were susceptible to vancomycin, metronidazole, chloramphenicol and rifampicin, while PCR-ribotypes 078, UCL 5a, UCL 36 and UCL 103 were associated with erythromycin resistance. The data suggest a wide dissemination of clones at hospitals and breeding-farms or a contamination at the slaughterhouse, but less probability of interspecies transmission. However, further highly discriminatory genotyping methods are necessary to elucidate interspecies and zoonotic transmission of C. difficile.

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.

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 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.

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.

Carriage and acquisition rates of Clostridium difficile in hospitalized horses, including molecular characterization, multilocus sequence typing and antimicrobial susceptibility of bacterial isolates.

Clostridium difficile has been identified as a significant agent of diarrhoea and enterocolitis in both foals and adult horses. Hospitalization, antibiotic therapy or changes in diet may contribute to the development of C. difficile infection. Horses admitted to a care unit are therefore at greater risk of being colonized. The aim of this study was to investigate the carriage of C. difficile in hospitalized horses and the possible influence of some risk factors in colonization. During a seven-month period, faecal samples and data relating the clinical history of horses admitted to a veterinary teaching hospital were collected. C. difficile isolates were characterized through toxin profiles, cytotoxicity activity, PCR-ribotyping, antimicrobial resistance and multilocus sequence typing (MLST). Ten isolates were obtained with a total of seven different PCR-ribotypes, including PCR-ribotype 014. Five of them were identified as toxinogenic. A high resistance to gentamicin, clindamycin and ceftiofur was found. MLST revealed four different sequencing types (ST), which included ST11, ST26, ST2 and ST15, and phylogenetic analysis showed that most of the isolates clustered in the same lineage. Clinical history suggests that horses frequently harbour toxigenic and non-toxigenic C. difficile and that in most cases they are colonized regardless of the reason for hospitalization; the development of diarrhoea is more unusual.

Presence of Clostridium difficile in pigs and cattle intestinal contents and carcass contamination at the slaughterhouse in Belgium.

The objective of this study was to evaluate the presence of Clostridium difficile in intestinal and carcass samples collected from pigs and cattle at a single slaughterhouse. C. difficile was isolated in 1% and 9.9% of the pig and cattle intestinal contents and in 7.9% and 7% of cattle and pig carcass samples respectively. A total of 19 different PCR-ribotypes were identified, among them types 078 and 014. Seven of 19 ribotypes correlated with the PCR-ribotypes involved in human C. difficile infections in Belgium. This study confirms that animals are carriers of C. difficile at slaughter and ribotypes are identical than those in humans, and that carcass contamination occurs inside the slaughterhouse.

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.

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.