Patient population
As shown in Figure 1, of 800 adult patients admitted to ICU during the study period, 115 patients developed diarrhea and 33 (28.70%) were identified as having a CDI. Twenty-five toxigenic C. difficile strains were also isolated from non-diarrhea patients, which were defined as CDC cases. The overall prevalence of CDIs and CDCs was 4.12% and 3.12%, respectively, all of which were healthcare facility-associated. Only one patient showed recurrence of infection, one patient transitioned from colonization to infection and two patients had infections of two different types. To assess the potential risk factors and clinical features, 66 non-CDI and 50 non-CDC patients were included as control groups. CDI and CDC patients had a median age of 54.15 and 62 years old, the proportion of men was 66.7% and 68%, and the number of days after admission when patients tested positive was 17.06±12.97 and 31.16±33.85 days, respectively. Neither age nor sex showed any significant difference between groups.
Clinical features and risk factors for ICU patients with CDI
As illustrated in Table 1, univariate analysis was conducted to show the differences between the CDI group and the controls with diarrhea in terms of clinical characteristics, diagnosis and treatment. The CDI group were more likely to suffer from fever (OR=6.786, 95% CI 2.634-17.483) (P value <0.001) and metabolic disorders (OR=3.28, 95% CI 1.363-7.893) (P <0.05) compared with the non-CDI group. CDI patients also displayed a larger number of comorbidities (P <0.05). Compared with the control group, patients with CDI more frequently received enteral feeding (78.8% versus 50%) (OR=3.714, 95% CI 1.416-9.74), antiviral drugs (15.2% versus 1.52%) (OR=11.607, 95% CI 1.296-103.948) and fluoroquinolone (21.2% versus 3%) (OR=8.615, 95% CI 1.678-44.247) during their hospitalization (P <0.05). Additionally, a larger proportion of CDI patients were administered more than one type of antibiotic drugs(P <0.05). To further assess the potential risk of CDI, multivariable logistic regression analysis was performed. The results showed that having a fever or metabolic disorder, or treatment with fluoroquinolone or combined antibiotics, were risk factors associated with the development of CDI among ICU patients. However, treatment with metronidazole was found to be a protective factor (OR=0.042, 95% CI 0.006-0.288, P=0.001).
Table 1 Univariate and multivariate analysis of the demographic, clinical characteristics, and risk factors in CDI groups
Characteristics
|
CDI group
(n=33)
|
non-CDI group
(n=66)
|
Univariate Analysis
|
Multivariable logistic regression Analysis
|
n(%)/mean±SD
|
n(%)/mean±SD
|
OR (95% CI)
|
P value
|
OR (95% CI)
|
P value
|
Male
|
22(66.7)
|
39(60)
|
0.722
(0.301-1.732)
|
0.465
|
|
|
Age(mean±SD)
|
54.15±20.89
|
58.97±14.87
|
-
|
0.242
|
|
|
Clinical features
|
Hospital duration (days)
(mean±SD)
|
35.39±27.61
|
30.08±33.11
|
-
|
0.429
|
|
|
Fever (≥ 38°C)
|
19(57.6)
|
11(16.7)
|
6.786
(2.634-17.483)
|
<0.001*
|
13.993
(3.292-59.472)
|
<0.001*
|
Leukocyte count (109 /L)
(mean±SD)
|
9.79±5.35
|
10.61±6.24
|
-
|
0.992
|
|
|
Serum albumin (g/L)
(mean±SD)
|
30.30±6.02
|
29.97±7.01
|
-
|
0.816
|
|
|
Serum creatinine rise>50%(μmol/L)
|
2(6.06)
|
12(18.18)
|
-
|
0.103
|
|
|
Mortality
|
6(18.2)
|
11(16.7)
|
1.111
(0.371-3.325)
|
0.851
|
|
|
Classification of primary diagnosis
|
Gastrointestinal
|
29(87.9)
|
55(83.8)
|
1.45
(0.424-4.959)
|
0.552
|
|
|
Respiratory
|
12(36.4)
|
26(39.4)
|
0.879
(0.37-2.086)
|
0.770
|
|
|
Cardiovascular
|
6(18.2)
|
14(21.2)
|
0.825
(0.285-2.39)
|
0.723
|
|
|
Renal
|
5(15.2)
|
17(25.8)
|
0.515
(0.171-1.546)
|
0.231
|
|
|
Neurologic
|
8(24.2)
|
9(13.6)
|
2.027
(0.701-5.862)
|
0.187
|
|
|
Metabolic disorders
|
22(66.7)
|
25(37.9)
|
3.28
(1.363-7.893)
|
0.007*
|
7.972
(1.767-35.971)
|
0.007*
|
NO. of comorbiditiesa
|
1-2
|
15(45.5)
|
43(65.2)
|
-
|
0.037*
|
|
|
3-4
|
16(48.5)
|
21(31.8)
|
≥5
|
2(6.1)
|
1(1.5)
|
Treatments and procedures
|
Surgical intervention
|
7(21.2)
|
15(22.7)
|
0.915
(0.332-2.523)
|
0.864
|
|
|
Enteral feeding
|
26(78.8)
|
33(50)
|
3.714
(1.416-9.74)
|
0.006*
|
|
|
PPI use
|
17(51.5)
|
43(65.2)
|
0.568
(0.243-1.330)
|
0.191
|
|
|
Antibiotics use
|
31(93.9)
|
57(86.4)
|
2.447
(0.497-12.042)
|
0.258
|
|
|
Antiviral drugs
|
5(15.2)
|
1(1.52)
|
11.607
(1.296-103.948)
|
0.007*
|
|
|
Antifungal agents
|
6(18.2)
|
6(9.1)
|
2.222
(0.657-7.522)
|
0.191
|
|
|
Cephalosporin
|
9(27.3)
|
26(29.4)
|
0.577
(0.232-1.435)
|
0.234
|
|
|
Fluoroquinolone
|
7(21.2)
|
2(3.0)
|
8.615
(1.678-44.247)
|
0.003*
|
42.696
(3.895-468.058)
|
0.002*
|
Carbapenem
|
24(72.7)
|
35(53.0)
|
2.362
(0.955-5.843)
|
0.060
|
|
|
Vancomycin
|
10(20.3)
|
13(19.7)
|
1.773
(0.68-4.624)
|
0.239
|
|
|
Metronidazole
|
5(15.2)
|
22(33.3)
|
0.357
(0.121-1.052)
|
0.056
|
0.042
(0.006-0.288)
|
0.001*
|
NO. of antibiotics receiveda
|
0
|
2(6.1)
|
9(13.6)
|
-
|
0.024*
|
2.856
(1.362-5.99)
|
0.005*
|
1-2
|
20(6.1)
|
48(72.7)
|
≥3
|
11(33.3)
|
9(13.6)
|
Numerical data are given as the mean ± SD, and categorical data are described as frequencies (percentages).
* P < 0.05; a The variables "No. of comorbidities" and “No. of antibiotics received” were made categorical, and the Cochran–Armitage trend test was used to analyze the differences in these variables between the two groups.
Clinical features and risk factors for ICU patients with CDC
For CDC patients, the median hospital stay was 62 days, significantly longer than that for non-CDC patients (P<0.05), which was further verified in the multivariable logistic regression model. The colonization of C. difficile did not cause a significant difference in the laboratory test indices including the laboratory leukocyte count, or serum albumin or creatinine levels. However, patients with respiratory or neurological disease were more likely to acquire C. difficile asymptomatically. The number of comorbidities was a potential risk factor for CDC patients (OR=36.509, 95% CI 2.602-512.183, P=0.08). As for the treatment procedure, surgical intervention, enteral feeding, antifungal agent usage, as well as carbapenem medication, were found more frequently in CDC patients (P<0.05). The multivariable model analysis showed that vancomycin was regarded as an independent risk factor (OR=18.168, 95% CI 1.036-318.503, P=0.047), whereas metronidazole use was a protective factor(OR=0.013, 95% CI 0-0.512, P=0.021) for C. difficile carriage (Table 2).
Table 2 Univariate and multivariate analysis of the demographic, clinical characteristics, and risk factors in CDC groups
Characteristics
|
CDI group
(n=33)
|
non-CDI group
(n=66)
|
Univariate Analysis
|
Multivariable logistic regression Analysis
|
n(%)/mean±SD
|
n(%)/mean±SD
|
OR (95% CI)
|
P value
|
OR (95% CI)
|
P value
|
Male
|
17(68.0)
|
31(62.0)
|
0.768
(0.278-2.121)
|
0.610
|
|
|
Age(mean±SD)
|
62±18.93
|
59.06±10.54
|
-
|
0.660
|
|
|
Clinical features
|
Hospital duration (days)
(mean±SD)
|
61.28±66.12
|
16.98±11.48
|
-
|
0.003*
|
1.137(1.05-1.23)
|
0.002*
|
Fever (≥ 38°C)
|
9(36.0)
|
7(14.0)
|
|
|
|
|
Leukocyte count (109 /L)
(mean±SD)
|
9.84±5.32
|
8.75±5.07
|
-
|
0.389
|
|
|
Serum albumin (g/L)
(mean±SD)
|
31.12±5.55
|
33.80±10.53
|
-
|
0.238
|
|
|
Serum creatinine rise>50%(μmol/L)
|
2(8.0)
|
5(10)
|
-
|
0.779
|
|
|
Mortality
|
3(12.0)
|
2(4.0)
|
3.273
(0.51-21.002)
|
0.190
|
|
|
Classification of primary diagnosis
|
Gastrointestinal
|
18(72.0)
|
30(60.0)
|
1.714
(0.606-4.852)
|
0.307
|
|
|
Respiratory
|
15(60.0)
|
19(38.0)
|
2.447
(0.916-6.541)
|
0.071
|
0.043(0.002-0.969)
|
0.048*
|
Cardiovascular
|
7(28.0)
|
18(36.0)
|
0.691
(0.243-1.969)
|
0.488
|
|
|
Renal
|
9(36.0)
|
15(30.0)
|
1.313
(0.475-3.626)
|
0.600
|
|
|
Neurologic
|
6(24.0)
|
3(6.0)
|
4.947
(1.121-21.838)
|
0.024*
|
|
|
Metabolic disorders
|
12(48.0)
|
26(52.0)
|
0.852
(0.326-2.227)
|
0.744
|
|
|
NO. of comorbidities
|
1-2
|
13(52.0)
|
33(66.0)
|
-
|
0.139
|
36.509
(2.602-512.183)
|
0.008*
|
3-4
|
9(36.0)
|
17(34.0)
|
≥5
|
3(12.0)
|
0(0)
|
Treatments and procedures
|
Surgical intervention
|
10(40.0)
|
4(8.0)
|
7.667
(2.094-28.068)
|
0.001*
|
|
|
Enteral feeding
|
16(64.0)
|
16(32.0)
|
3.778
(1.376-10.372)
|
0.008*
|
|
|
PPI use
|
8(32.0)
|
25(50.0)
|
0.471
(0.172-1.288)
|
0.139
|
|
|
Antibiotics use
|
24(96.0)
|
41(82.0)
|
5.268
(0.628-44.178)
|
0.093
|
|
|
Antiviral drugs
|
2(8.0)
|
3(6.0)
|
1.362
(0.213-8.729)
|
0.743
|
|
|
Antifungal agents
|
7(28.0)
|
4(8.0)
|
4.472
(1.166-17.146)
|
0.021*
|
|
|
Cephalosporin
|
9(36.0)
|
24(48.0)
|
0.609
(0.227-1.636)
|
0.324
|
|
|
Fluoroquinolone
|
5(20.0)
|
8(16.0
|
1.313
(0.381-4.525)
|
0.666
|
|
|
Carbapenem
|
18(70.0)
|
21(42.0)
|
3.551
(1.258-10.027)
|
0.014*
|
|
|
Vancomycin
|
12(48.0)
|
7(14.0)
|
5.67
(1.851-17.374)
|
0.001*
|
18.168(1.036-318.503)
|
0.047*
|
Metronidazole
|
2(8.0)
|
12(24.0)
|
0.275
(0.056-1.342)
|
0.094
|
0.013
(0-0.512)
|
0.021*
|
NO. of antibiotics received
|
0
|
1(4.0)
|
9(18.0)
|
-
|
0.076
|
|
|
1-2
|
16(64.0)
|
33(66.0)
|
≥3
|
8(32.0)
|
8(16.0)
|
Numerical data are given as the mean ± SD, and categorical data are described as frequencies (percentages).
* P < 0.05; a The variables "No. of comorbidities" and “No. of antibiotics received” were made categorical, and the Cochran–Armitage trend test was used to analyze the differences in these variables between the two groups.
Molecular characteristics of C. difficile
The toxin type was detected among 58 Clostridioides difficile strains isolated from CDI and CDC patients, and 34 (58.6%) were A+B+ (positive for both tcdA and tcdB) and 24 (41.3%) were A˗B+ (negative for tcdA and positive for tcdB). As for the two defined groups, 20 (60.6%) strains were A+B+ and 13 (39.4%) strains were A˗B+ in the CDI group, and 14 (56%) strains were A+B+ and 11 (44%) strains were A˗B+ in the CDC group.
Then, MLST was performed on these strains. In total, 18 sequence types (STs) were identified. In the CDI group, ST2, ST81, ST54 and ST3 were the major STs constituting 19%, 15%, 12% and 12% of strains, respectively. In the CDC group, ST81, ST35, ST37 and ST54 were the dominant types accounting for 20%, 12%, 12% and 12% of strains, respectively, as shown in Figure 2.
Based on the STs of strains, a map was constructed to compare the temporospatial relationship for the same STs from two groups during the study period, as shown in Figure 3. Two overlaps were detected within the CDI group in ST2 and one overlap was detected between the CDI and CDC groups in ST103. No overlaps were detected among other STs.