Asymptomatic Clostridium difficile colonisation and onward transmission.

INTRODUCTION: Combined genotyping/whole genome sequencing and epidemiological data suggest that in endemic settings only a minority of Clostridium difficile infection, CDI, is acquired from other cases. Asymptomatic patients are a potential source for many unexplained cases.
METHODS: We prospectively screened a cohort of medical inpatients in a UK teaching hospital for asymptomatic C. difficile carriage using stool culture. Electronic and questionnaire data were used to determine risk factors for asymptomatic carriage by logistic regression. Carriage isolates were compared with all hospital/community CDI cases from the same geographic region, from 12 months before the study to 3 months after, using whole genome sequencing and hospital admission data, assessing particularly for evidence of onward transmission from asymptomatic cases.
RESULTS: Of 227 participants recruited, 132 provided ≥1 stool samples for testing. 18 participants were culture-positive for C. difficile, 14/132(11%) on their first sample. Independent risk factors for asymptomatic carriage were patient reported loose/frequent stool (but not meeting CDI criteria of ≥3 unformed stools in 24 hours), previous overnight hospital stay within 6 months, and steroid/immunosuppressant medication in the last 6 months (all p≤0.02). Surprisingly antibiotic exposure in the last 6 months was independently associated with decreased risk of carriage (p = 0.005). The same risk factors were identified excluding participants reporting frequent/loose stool. 13/18(72%) asymptomatically colonised patients carried toxigenic strains from common disease-causing lineages found in cases. Several plausible transmission events to asymptomatic carriers were identified, but in this relatively small study no clear evidence of onward transmission from an asymptomatic case was seen.
CONCLUSIONS: Transmission events from any one asymptomatic carrier are likely to be relatively rare, but as asymptomatic carriage is common, it may still be an important source of CDI, which could be quantified in larger studies. Risk factors established for asymptomatic carriage may help identify patients for inclusion in such studies.

Introduction

Traditionally most Clostridium difficile infection (CDI) cases have been thought to result from healthcare-based exposure to other cases [1] and this forms the basis for many prevention measures [2]. However, combined genotyping and hospital admission data from two European cohorts suggest that, in these endemic settings with appropriate infection control, contact with symptomatic cases only accounts for the minority of disease [3], [4]. Applying the greater discriminatory power of bacterial whole genome sequencing in one of these cohorts demonstrates the majority of cases over the 3 year study period are sufficiently genetically diverse that they cannot be related by direct or indirect transmission irrespective of the route of transmission [5].

Data from previous studies suggest C. difficile is typically acquired shortly before onset of disease [6], with long term carriage protective against CDI [7], [8]. Therefore, although C. difficile is widely found in the environment [9], recent exposure to asymptomatic individuals, particularly in a healthcare setting, represents a compelling potential source for many currently unexplained CDI cases and a potential area for intervention. Between 4% and 15% of healthy adults may be asymptomatically colonised [10], [11]. In hospitalised adults, asymptomatic carriage rates reported in a large Canadian study were 184/4143 (4.4%) at admission with a further 123 (3.0%) becoming asymptomatically colonized during their hospital stay. However the wards included were selected to be a mix of previously high and low CDI incidence wards, and therefore, despite the considerable size of the study, generalising the findings is difficult [6]. Five earlier studies, with a total of 1755 patients across a range of specialties, report asymptomatic carriage rates at admission of 6–11%, and acquisition rates of 4–21%, with more than 63% of these patients remaining asymptomatic [12]. Colonisation in young children is common, with rates ∼35% in the first year of life falling to ∼15% by 1–8 years [13]. High colonisation rates are also described in long-term care facilities, 51% in the context of an outbreak [14], and 4–20% in endemic settings [15], [16].

Compared with non-colonised patients, asymptomatic patients have higher rates of skin and environmental contamination (but less than that associated with symptomatic cases) [14], [17], [18], and C. difficile can be recovered from investigators’ hands after contact with colonized individuals [14]. Estimates of the extent of onward transmission within hospitals attributable to asymptomatic carriers vary considerably [19], [20]. In one study, nosocomial acquisition of a new C. difficile strain was preceded by initial introduction of the strain on to a ward by an asymptomatic carrier in 16/19 (84%) instances [19]. In another examining possible sources for recently diagnosed cases, 5/12 symptomatic contacts had a strain matching the new case compared to 1/19 asymptomatic contacts [20]. However these findings are based on relatively old studies and genotyping methods. Therefore, while onward transmission from asymptomatically colonised patients is clearly possible, its current relative importance as a source of CDI is unclear. We therefore prospectively screened a cohort of medical inpatients for asymptomatic carriage of C. difficile. Carriage isolates were then compared to all subsequent CDI cases in the same geographic region over the following 3 months for evidence of onward transmission from asymptomatic cases.

Methods

Setting and Participants

This study was performed at the Oxford University Hospitals NHS Trust, OUH. The OUH consists of 1600 beds across 4 hospital sites in the English county of Oxfordshire (population ∼600000). The hospitals provide all acute care to the population, >90% of all hospital services, and all diagnostic testing for C. difficile for hospital and community samples from the county.

All patients aged over 18 years admitted to a study ward were eligible for inclusion. The study was conducted on acute general medicine and geratology wards, as these specialities had the greatest historic CDI incidence. The choice of high prevalence wards was intended to provide the maximum number of CDI cases on the same ward as asymptomatic carriers, in order to investigate the potential for carrier to case transmissions. Patients diagnosed with CDI in the last 28 days, or with a current clinical suspicion of CDI (≥3 unformed stools in a 24 hour period) were excluded from this specific study (but samples/sequences from these cases were available for comparison with study participants, see below).

Recruitment was rotated around 8 study wards on a weekly to fortnightly basis (between 02 February 2012 and 10 June 2012) to reduce the nursing burden of sending stool samples from patients. Following informed consent (or the advice of an appropriate consultee in the case of patients without capacity) patients were recruited within 24 hours of ward admission where possible (however, no upper limit was set for the time between admission and enrolment).

Samples

Patients were asked provide a sample of their first stool after enrolment, and then a sample at approximately 3 day intervals until discharge, and where possible a sample shortly prior to discharge.

Samples were cultured in weekly batches following the method of [21], which is very similar to that used in largest study of asymptomatic carriage to date [6]. Samples with colonies exhibiting a characteristic appearance, odour, and fluorescence consistent with C. difficile underwent whole genome sequencing as described in [5]. Briefly, indexed pools of 96 samples were sequenced using the Illumina HiSeq 2000 platform (Illumina Inc, San Diego, CA) generating 100 base pair paired-end reads. Reads were mapped with Stampy [22] to the C. difficile reference genome 630 (Genbank: AM180355.1) [23]. Samples were compared using single nucleotide variants (SNVs) identified with Samtools [24] after quality filtering. For each sequence the presence or absence of toxin genes and an in silico multilocus sequence type, MLST, was determined from de novo assemblies constructed with Velvet [25] using BLAST.

CDI Cases for Comparison

All Oxfordshire hospital and community clinical CDI cases from April 2011 to August 2012 inclusive were considered for comparison. During this time all hospital inpatients with ≥3 unformed stools in 24 hours underwent C. difficile testing. Between April 2011 and March 2012 samples sent to the OUH microbiology laboratory underwent toxin EIA-testing, and EIA-positive samples were cultured. Between April 2012 and August 2012 samples were screened with a GDH test, with a follow up confirmatory EIA test in GDH-positive samples. All GDH-positive samples were cultured, regardless of the subsequent EIA result. This enabled potential C. difficile ‘excretors’ carrying C. difficile, but not currently producing toxin, to be included. Between April 2011 and August 2012 a total of 505 symptomatic C. difficile culture-positive samples were identified, 481 (95%) of which were successfully whole genome sequenced and available for comparison. A further 137 sequences were available from a C. difficile diagnostic study, conducted between April 2011 and September 2011, that were culture-positive, but EIA-negative [26].

Epidemiological Data

Consent was obtained for use of hospital electronic data, including participant demographics (age, sex, postcode district (first 3–4 characters of the postcode)), hospital admission, discharge and ward movement data within the OUH, and previous and future routine C. difficile testing). Additional data on potential risks factors for C. difficile carriage were collected from patients using a questionnaire and hospital records (see and ).

Table 1

Comparison of characteristics between participants returning and not returning at least one stool sample in 199 participants with questionnaire data.

Risk factor	Frequency in those not returning samples	Frequency in those returning samples	p value
Current diarrhoea, but not meeting CDI criteria	8/86 (9%)	22/111 (20%)	0.05
Nursing home, residential home, or institutional resident	8/87 (9%)	6/112 (5%)	0.40
Inflammatory bowel disease	0/86 (0%)	1/112 (1%)	1.00
Chronic kidney disease	17/86 (20%)	24/112 (21%)	0.86
Non-haematological malignancy	4/86 (5%)	17/112 (15%)	0.02
Haematological malignancy	2/86 (2%)	5/112 (4%)	0.70
Gastrointestinal surgery in current admission	1/86 (1%)	5/112 (4%)	0.24
Nasogastric tube in place	1/86 (1%)	3/112 (3%)	0.63
Overnight hospital stay in last 6 months	41/84 (49%)	43/111 (39%)	0.19
Outpatient hospital attendance in last 6 months	24/84 (29%)	33/111 (30%)	0.88
GP visits in last 6 months, n	87	112	0.30
0	4 (5%)	9 (8%)
1–2	31 (36%)	36 (32%)
3–9	28 (32%)	46 (41%)
10+	24 (28%)	21 (19%)
Visitor to hospital, nursing home, resident home	5/87 (6%)	3/111 (3%)	0.30
Work in healthcare	2/87 (2%)	3/111 (3%)	1.00
Healthcare worker in household	3/87 (3%)	9/111 (8%)	0.23
Previous C. difficile infection	0/87 (0%)	5/111 (5%)	0.07
Previous contact with a C. difficile infection case	3/87 (3%)	2/111 (2%)	0.66
Antibiotics in last 6 months	65/87 (75%)	90/112 (80%)	0.39
Steroids/Immunosuppressant in last 6 months	25/87 (29%)	28/112 (25%)	0.63
Gastric acid suppressant in last 6 months	25/87 (29%)	54/112 (48%)	0.006
Household pet	25/86 (29%)	27/111 (24%)	0.52
Physical contact with children ≥1 per month	49/86 (57%)	54/110 (49%)	0.31
Vegetarian diet	5/75 (6%)	7/111 (6%)	1.00
Foreign travel in last 6 months	3/87 (3%)	4/112 (4%)	1.00
Current smoker	10/87 (11%)	11/112 (10%)	0.82

Exact p values shown. Not all participants could answer all study questionnaire questions, as indicated in the dominator given in each line.

Table 2

Univariate risk factors for asymptomatic carriage of C. difficile.

Risk factor	C. difficile carriers(% total)	Non-carriers(% total)	Odds ratio, carriers:non-carriers	95% CI	p value
Current diarrhoea, but not meeting CDI criteria	5 (50%)	17 (17%)	4.94	(1.29, 19.0)	0.02
Nursing or residential home resident	2 (20%)	4 (4%)	6.13	(0.97, 38.7)	0.08
Chronic kidney disease	2 (20%)	22 (22%)	1.10	(0.21, 5.56)	0.90
Non-haematological malignancy	3 (30%)	14 (14%)	2.69	(0.62, 11.66)	0.21
Overnight hospital stay in last 6 months	6 (60%)	37 (36%)	2.59	(0.68, 9,79)	0.15
Time in hospital during current admission priorto enrolment, odds ratio per day*			1.00	(0.95, 1.05)	0.99
Outpatient hospital attendance in last 6 months	2 (20%)	31 (31%)	0.56	(0.11, 2.81)	0.47
GP visits in last 6 months
0	2 (20%)	7 (7%)	1.77	(0.28, 11.1)	0.23
1–2	5 (50%)	31 (30%)	1
3–9	2 (20%)	44 (43%)	0.28	(0.05, 1.55)
10+	1 (10%)	20 (20%)	0.41	(0.03, 2.85)
Healthcare worker in household	1 (10%)	8 (8%)	1.29	(0.14, 11.52)	0.82
Antibiotics in last 6 months	6 (60%)	84 (82%)	0.32	(0.08, 1.25)	0.12
Steroids/Immunosuppressant in last 6 months	5 (50%)	23 (23%)	3.43	(0.91, 12.9)	0.07
Gastric acid suppressant in last 6 months	7 (70%)	47 (46%)	2.73	(0.67, 11.2)	0.14
Household pet	3 (33%)	24 (24%)	1.62	(0.38, 7.00)	0.52
Physical contact with children ≥1 per month	3 (33%)	51 (51%)	0.49	(0.11, 2.07)	0.32
Current smoker	1 (10%)	10 (10%)	1.02	(0.11, 8.92)	0.98

There were no C. difficile carriers with the following risk factors: inflammatory bowel disease (present in 1/112 (1%) participants), haematological malignancy (5/112 (4%)), gastrointestinal surgery (5/112 (4%) with surgery in current admission, 12/112 (11%) with past surgery), current nasogastric tube placement (3/112 (3%)), previous CDI (5/112 (4%)), contact with a CDI case (2/112 (2%)), vegetarian diet (7/112 (6%)), and overseas travel (4/112 (4%)). *Given the study design, most participants spent relatively short amounts of time in hospital prior to enrolment, median (IQR) 2 (1–6) days.

Analysis

Data obtained were used to estimate the point prevalence of asymptomatic carriage of C. difficile. Complete assessment of prevalence at admission and discharge, and therefore the incidence of acquisition, was not possible given the time delay between many participants’ recruitment and first bowel motion, and similarly relatively few participants providing more than one sample (see results and discussion).

Logistic regression was used to assess associations between the presence of C. difficile carriage in each participant’s first stool sample and risk factors for asymptomatic carriage. Univariate and multivariate models were fitted on cases with complete questionnaire data (n = 110/112 with any questionnaire data), with independent risk factors identified using backwards selection with the Akaike information criterion starting from a model including all variables with univariate p<0.20 [27], and then the final model re-fitted on all participants with complete data for the final variables included (n = 111). Because of the small size of the study we consider p<0.15 as marginal evidence of an effect and p<0.05 as significant evidence of an effect.

Whole genome sequences obtained from asymptomatic carriers were compared to CDI cases’ sequences obtained from 1 year prior to the study, during the study and for 3 months afterwards. Ward movements of cases and carriers sharing closely related C. difficile genomes were analysed for evidence of possible transmission events.

Ethics

This study was approved by the Oxford C Research Ethics Committee (11/SC/0197). Samples from 20 asymptomatic study ward inpatients collected as part of routine infection control surveillance are also included in the analysis (no questionnaire data). Use of routinely collected samples and data, including from symptomatic patients, for the purposes of transmission analyses was approved by the Berkshire Research Ethics Committee (10/H0505/83) and National Information Governance Board (8–05(e)/2010) without requiring individual patient consent.

Data Sharing

The sequences reported in this paper have been deposited in the European Nucleotide Archive Sequence Read Archive under study accession number <<