Deirdre A. Collins

Epidemiology of Clostridium difficile infection in Asia

While Clostridium difficile infection (CDI) has come to prominence as major epidemics have occurred in North America and Europe over the recent decade, awareness and surveillance of CDI in Asia have remained poor. Limited studies performed throughout Asia indicate that CDI is also a significant nosocomial pathogen in this region, but the true prevalence of CDI remains unknown. A lack of regulated antibiotic use in many Asian countries suggests that the prevalence of CDI may be comparatively high. Molecular studies indicate that ribotypes 027 and 078, which have caused significant outbreaks in other regions of the world, are rare in Asia. However, variant toxin A-negative/toxin B-positive strains of ribotype 017 have caused epidemics across several Asian countries. Ribotype smz/018 has caused widespread disease across Japan over the last decade and more recently emerged in Korea. This review summarises current knowledge on CDI in Asian countries.

Epidemiology of Clostridium difficile infection in two tertiary-care hospitals in Perth, Western Australia: a cross-sectional study

The epidemiology of Clostridium difficile infection (CDI) has changed over time and between countries. It is therefore essential to monitor the characteristics of patients at risk of infection and the circulating strains to recognize local and global trends, and improve patient management. From December 2011 to May 2012 we conducted a prospective, observational epidemiological study of patients with laboratory-confirmed CDI at two tertiary teaching hospitals in Perth, Western Australia to determine CDI incidence and risk factors in an Australian setting. The incidence of CDI varied from 5.2 to 8.1 cases/10 000 occupied bed days (OBDs) at one hospital and from 3.9 to 16.3/10 000 OBDs at the second hospital. In total, 80 patients with laboratory-confirmed CDI met eligibility criteria and consented to be in the study. More than half (53.8%) had hospital-onset disease, 28.8% had community-onset and healthcare facility-associated disease and 7.5% were community-associated infections according to the definitions used. Severe CDI was observed in 40.0% of these cases but the 30-day mortality rate for all cases was only 2.5%. Besides a shorter length of stay among cases of community-onset CDI, no characteristics were identified that were significantly associated with community-onset or severe CDI. From 70 isolates, 34 different ribotypes were identified. The predominant ribotypes were 014 (24.3%), 020 (5.7%), 056 (5.7%) and 070 (5.7%). Whereas this study suggests that the characteristics of CDI cases in Australia are not markedly different from those in other developed countries, the increase in CDI rate observed emphasizes the importance of surveillance.

Genome analysis of Clostridium difficile PCR ribotype 014 lineage in Australian pigs and humans reveals a diverse genetic repertoire and signatures of long-range interspecies transmission

Clostridium difficile PCR ribotype (RT) 014 is well-established in both human and porcine populations in Australia, raising the possibility that C. difficile infection (CDI) may have a zoonotic or foodborne etiology. Here, whole genome sequencing and high-resolution core genome phylogenetics were performed on a contemporaneous collection of 40 Australian RT014 isolates of human and porcine origin. Phylogenies based on MLST (7 loci, STs 2, 13, and 49) and core orthologous genes (1260 loci) showed clustering of human and porcine strains indicative of very recent shared ancestry. Core genome single nucleotide variant (SNV) analysis found 42% of human strains showed a clonal relationship (separated by ≤2 SNVs in their core genome) with one or more porcine strains, consistent with recent inter-host transmission. Clones were spread over a vast geographic area with 50% of the human cases occurring without recent healthcare exposure. These findings suggest a persistent community reservoir with long-range dissemination, potentially due to agricultural recycling of piggery effluent. We also provide the first pan-genome analysis for this lineage, characterizing its resistome, prophage content, and in silico virulence potential. The RT014 is defined by a large “open” pan-genome (7587 genes) comprising a core genome of 2296 genes (30.3% of the total gene repertoire) and an accessory genome of 5291 genes. Antimicrobial resistance genotypes and phenotypes varied across host populations and ST lineages and were characterized by resistance to tetracycline [tetM, tetA(P), tetB(P) and tetW], clindamycin/erythromycin (ermB), and aminoglycosides (aph3-III-Sat4A-ant6-Ia). Resistance was mediated by clinically important mobile genetic elements, most notably Tn6194 (harboring ermB) and a novel variant of Tn5397 (harboring tetM). Numerous clinically important prophages (Siphoviridae and Myoviridae) were identified as well as an uncommon accessory gene regulator locus (agr3). Conservation in the pathogenicity locus and S-layer correlated with ST affiliation, further extending the concept of clonal C. difficile lineages. This study provides novel insights on the genetic variability and strain relatedness of C. difficile RT014, a lineage of emerging One Health importance. Ongoing molecular and genomic surveillance of strains in humans, animals, food, and the environment is imperative to identify opportunities to reduce the overall CDI burden.

Molecular methods for detecting and typing of Clostridium difficile

Summary Since the early 2000s, Clostridium difficile has emerged as a major international pathogen. Recently, strains of C. difficile in circulation appear to be changing, with greater diversity, leading to challenges for diagnostics and surveillance. Currently molecular diagnostic methods are favoured for their high sensitivity and rapid processing times; however, a number of issues still exist with molecular tests, in particular high cost, low clinical specificity and failure to detect some variant C. difficile strains. Molecular typing methods are used to determine the continually evolving epidemiology of C. difficile infection. Typing methods including PCR ribotyping and pulsed field gel electrophoresis are currently popular in Europe and North America, respectively, while high-throughput next-generation sequencing is likely to become more widely used in years to come. This review discusses current molecular detection and typing techniques for C. difficile.

The emergence of community-onset Clostridium difficile infection in a tertiary hospital in Singapore: A cause for concern

Increasing rates of Clostridium difficile infection (CDI) among those without traditional risk factors have been reported mainly in Europe and North America. Here we describe the epidemiology, clinical features and ribotypes of CDI at National University Hospital (NUH), a 1000-bed tertiary care hospital in Singapore, from December 2011 to May 2012. All laboratory-confirmed CDI cases ≥21 years old who gave informed consent were included. Clinical data were collected prospectively and participants underwent an interviewer-administered questionnaire. Cases were classified by healthcare facility exposure and severity according to the SHEA guidelines. Included cases were also subjected to PCR and were classified by ribotype. In total, 66 patients participated in the study, of which 33 (50.0%) were healthcare-facility-associated hospital onset (HCFA-HO). Of the 33 community-onset (CO) cases, 14 (42.4%) were HCFA-CO, 10 (30.3%) were indeterminate and 9 (27.3%) were community-associated (CA). Of the CA cases, a majority (90.9%) had prior exposure to a healthcare facility within the last 12 weeks. Clinical characteristics, exposures and outcomes were not different between HO-CDI and CO-CDI. Diagnosis was delayed in CO-CDI compared with HO-CDI (4 days vs. 1 day; P=0.014). There was no difference in distribution of ribotypes between CO-CDI and HO-CDI, with 053 being most prevalent in both groups. CO-CDI increasingly contributes to the burden of CDI in NUH. This may reflect a trend in other parts of Asia. Healthcare professionals should be aware of the possible role of outpatient healthcare environments to CDI risk and thus extend control measures to outpatient settings.

Routine detection of Clostridium difficile in Western Australia.

Despite increasing infection rates, Clostridium difficile is not currently routinely tested for in all diarrhoeal faecal specimens in Australia. In July 2014, all diarrhoeal specimens submitted to a diagnostic laboratory in Western Australia were surveyed to determine the true prevalence of C. difficile. In total, 1010 diarrhoeal non-duplicate faecal specimens were received during the month. Testing for C. difficile was requested, or the criteria for a C. difficile investigation were met, for 678 specimens which were investigated by PCR for the tcdB gene using the BD MAX platform, followed by toxigenic culture on PCR-positive samples. The remaining 332 specimens, with either no C. difficile test request or the criteria for a C. difficile investigation were not met, were examined by toxigenic culture. All isolates were PCR ribotyped. C. difficile was the most commonly detected diarrhoeal pathogen among all specimens. The overall prevalence of C. difficile in all 1010 specimens was 6.4%; 7.2% in the routinely tested group, and 4.8% in the non-requested group. The proportion of non-requested positive detections among all cases was 24.6%. Community-onset infection was present in 50.8% of all cases. The median age of all CDI cases was 60.0 years and the age range in CDI patients in the routine group was 0.6-96.6 years (median 72.7 years), compared to 0.2-2.3 years (median 0.8 years) in the non-requested group. The most common ribotype (RT) found was RT 014/020 (34.1% in the routine group, 43.8% in the non-requested group), followed by RTs 002, 056, 005 and 018. While the routine testing group and the non-requested group differed markedly in age and patient classification, C. difficile was the most common cause of diarrhoea in hospitals and the community in Western Australia. The significance of finding C. difficile in the community paediatric population requires further study.

Incorrect diagnosis of Clostridium difficile infection in a university hospital in Japan.

Physicians often fail to suspect Clostridium difficile infection (CDI) and many microbiology laboratories use suboptimal diagnostic techniques. To estimate the extent of and reasons for incorrect diagnosis of CDI in Japan, we investigated toxigenic C. difficile isolated from all stool culture samples and clinical course. Over a 12-month period in 2010, all stool culture samples (n = 975) submitted from inpatients in a university hospital in Japan were cultured for C. difficile and routine microbiological testing was conducted. In total, 177 C. difficile isolates were recovered, and 127 isolates were toxigenic. Among the toxin-A-positive/toxin-B-positive isolates, 12 were also positive for the binary toxin gene. However, clinically important ribotypes, such as 027 and 078, were not identified. A total of 58 (45.7%) cases with toxigenic C. difficile had unformed stool, and the incidence CDI was 1.6 cases per 10,000 patient-days. Of these 58 cases, 40 were not diagnosed in routine testing due to a lack of clinical suspicion (24.1%, 14/58) or a negative C. difficile toxin assay result (44.8%, 26/58). A stool toxin assay was performed in 54 patients (78.2%, 54/69) who did not have unformed stool. The present study demonstrated that a significant number of CDI cases in Japan might be overlooked or misdiagnosed in clinical practice due to a lack of clinical suspicion and limitations of microbiological testing for CDI in Japan. Providing education to promote awareness of CDI among physicians is important to improve the accuracy of diagnosis in Japan.

High Nasopharyngeal Carriage of Non-Vaccine Serotypes in Western Australian Aboriginal People Following 10 Years of Pneumococcal Conjugate Vaccination

Background Invasive pneumococcal disease (IPD) continues to occur at high rates among Australian Aboriginal people. The seven-valent pneumococcal conjugate vaccine (7vPCV) was given in a 2-4-6-month schedule from 2001, with a 23-valent pneumococcal polysaccharide vaccine (23vPPV) booster at 18 months, and replaced with 13vPCV in July 2011. Since carriage surveillance can supplement IPD surveillance, we have monitored pneumococcal carriage in western Australia (WA) since 2008 to assess the impact of the 10-year 7vPCV program. Methods We collected 1,500 nasopharyngeal specimens from Aboriginal people living in varied regions of WA from August 2008 until June 2011. Specimens were cultured on selective media. Pneumococcal isolates were serotyped by the quellung reaction. Results Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were carried by 71.9%, 63.2% and 63.3% respectively of children <5 years of age, and 34.6%, 22.4% and 27.2% of people ≥5 years. Of 43 pneumococcal serotypes identified, the most common were 19A, 16F and 6C in children <5 years, and 15B, 34 and 22F in older people. 7vPCV serotypes accounted for 14.5% of all serotypeable isolates, 13vPCV for 32.4% and 23vPPV for 49.9%, with little variation across all age groups. Serotypes 1 and 12F were rarely identified, despite causing recent IPD outbreaks in WA. Complete penicillin resistance (MIC ≥2µg/ml) was found in 1.6% of serotype 19A (5.2%), 19F (4.9%) and 16F (3.2%) isolates and reduced penicillin susceptibility (MIC ≥0.125µg/ml) in 24.9% of isolates, particularly 19F (92.7%), 19A (41.3%), 16F (29.0%). Multi-resistance to cotrimoxazole, tetracycline and erythromycin was found in 83.0% of 23F isolates. Among non-serotypeable isolates 76.0% had reduced susceptibility and 4.0% showed complete resistance to penicillin. Conclusions Ten years after introduction of 7vPCV for Aboriginal Australian children, 7vPCV serotypes account for a small proportion of carried pneumococci. A large proportion of circulating serotypes are not covered by any currently licensed vaccine.

Prevalence and molecular epidemiology of Clostridium difficile infection in Indonesia

Clostridium difficile has not been studied in detail in Asia, particularly Southeast Asia. We thus performed a prevalence study across four hospitals in Central Java province, Indonesia. Stool samples were collected from patients with diarrhoea and tested by enzyme immunoassay for glutamate dehydrogenase (GDH) and toxin A/B (C DIFF QUIK CHEK COMPLETE, TechLab). Specimens were cultured and molecular typing was performed. In total, 340 samples were tested, of which 70 (20.6%) were GDH positive, with toxin detected in 19 (5.6%). Toxigenic C. difficile was isolated from 37 specimens (10.9%), while a further 36 (10.6%) nontoxigenic isolates were identified. The most common strain was ribotype 017 (24.3% of 74 isolates), followed by nontoxigenic types QX 224 (9.5%), and QX 238 and QX 108 (both 8.1%). The high prevalence of C. difficile highlights a need for ongoing surveillance of C. difficile infection in Indonesia.

Prevalence of Clostridium difficile infection and colonization in a tertiary hospital and elderly community of North-Eastern Peninsular Malaysia

Little is known about Clostridium difficile infection (CDI) in Asia. The aims of our study were to explore (i) the prevalence, risk factors and molecular epidemiology of CDI and colonization in a tertiary academic hospital in North-Eastern Peninsular Malaysia; (ii) the rate of carriage of C. difficile among the elderly in the region; (iii) the awareness level of this infection among the hospital staffs and students. For stool samples collected from hospital inpatients with diarrhea (n = 76) and healthy community members (n = 138), C. difficile antigen and toxins were tested by enzyme immunoassay. Stool samples were subsequently analyzed by culture and molecular detection of toxin genes, and PCR ribotyping of isolates. To examine awareness among hospital staff and students, participants were asked to complete a self-administered questionnaire. For the hospital and community studies, the prevalence of non-toxigenic C. difficile colonization was 16% and 2%, respectively. The prevalence of CDI among hospital inpatients with diarrhea was 13%. Out of 22 C. difficile strains from hospital inpatients, the toxigenic ribotypes 043 and 017 were most common (both 14%). In univariate analysis, C. difficile colonization in hospital inpatients was significantly associated with greater duration of hospitalization and use of penicillin (both P < 0·05). Absence of these factors was a possible reason for low colonization in the community. Only 3% of 154 respondents answered all questions correctly in the awareness survey. C. difficile colonization is prevalent in a Malaysian hospital setting but not in the elderly community with little or no contact with hospitals. Awareness of CDI is alarmingly poor.