The clinical characteristics of the patients infected with S. Enteritidis
Recently, 6 patients from one company visited our department, they all complained of fever (body temperatures ranged from 38.5℃ to 39.2℃), watery stool (Bristol type 7, defection frequency ranged from 3 times/day to 10 times/day), nausea, abdominal pain, chills and fatigue; some reported vomiting (1 out of 6, 16.7%), lower back pain (2/6, 33.3%), headache (2/6, 33.3%) and knee pain (2/6, 33.3%) (Table 1). They were all males aged between 18 and 23 years.
Table 1
The clinicalpathological variables of the patients when they were admitted.
|
Symptoms
|
Patient
|
Temperature (℃)
|
Defection frequency (times/day)
|
Stool Bristol type
|
Nausea
|
Abdominal pain
|
Chills
|
Fatigue
|
Vomit
|
Lower back pain
|
Headache
|
Knee pain
|
Case 1
|
39.1
|
5
|
7
|
+
|
+
|
+
|
+
|
-
|
+
|
+
|
+
|
Case 2
|
38.8
|
3
|
7
|
+
|
+
|
+
|
+
|
+
|
-
|
-
|
-
|
Case 3
|
39
|
8
|
7
|
+
|
+
|
+
|
+
|
-
|
-
|
-
|
-
|
Case 4
|
39
|
4
|
7
|
+
|
+
|
+
|
+
|
-
|
-
|
-
|
-
|
Case 5
|
38
|
4
|
7
|
+
|
+
|
+
|
+
|
-
|
+
|
+
|
+
|
Case 6
|
38.8
|
10
|
7
|
+
|
+
|
+
|
+
|
-
|
-
|
-
|
-
|
|
Blood routine test
|
Coagulation function
|
|
|
Inflammation
|
|
Patient
|
WBC
|
Neutrophil
|
Lymphocyte
|
Hb
|
Platelet
|
PT
|
APTT
|
Fibrinogen
|
D-dimer
|
CRP
|
|
(3.5–9.5*10^9/L)
|
(40–75%)
|
(20–50%)
|
(130–175 g/L)
|
(125–350 *10^9/L)
|
(9.4–12.5 s)
|
(25.1–36.5 s)
|
(2–4 g/L)
|
(0-299 mg/ml)
|
(0–5 mg/L)
|
|
Case 1
|
13.75
|
91.9
|
4.1
|
144
|
194
|
14.1
|
35.9
|
2.17
|
512
|
41.9
|
|
Case 2
|
8.37
|
86.7
|
9.2
|
153
|
172
|
16.5
|
27.7
|
2.21
|
428
|
7.2
|
|
Case 3
|
15.72
|
91.3
|
4.8
|
154
|
178
|
17.3
|
17.8
|
2.59
|
479
|
73.8
|
|
Case 4
|
11.54
|
88.5
|
6.3
|
153
|
161
|
13.2
|
31.8
|
2.06
|
422
|
14.3
|
|
Case 5
|
7.1
|
83.2
|
11.9
|
153
|
187
|
12.7
|
36.3
|
2.18
|
384
|
23.2
|
|
Case 6
|
10.03
|
78.4
|
14.5
|
163
|
179
|
13.1
|
34.6
|
2.21
|
296
|
8.3
|
|
|
Liver function
|
Renal function
|
|
|
Patient
|
ALT
|
AST
|
Albumin
|
Prealbumin
|
ALP
|
GGT
|
Total bile acid
|
Cr
|
BUN
|
Serum iron
|
|
(9–50 U/L)
|
(8–40 U/L)
|
(40–55 g/L)
|
(250–400 g/L)
|
(40–150 U/L)
|
(11–50 U/L)
|
(<10 µmol/L)
|
(62–115 µmol/L)
|
(2.9–8.2 mmol/L)
|
(10.6–36.7 µmol/L)
|
|
Case 1
|
15.6
|
21.8
|
48.6
|
218
|
63.3
|
15.2
|
1
|
89.6
|
4.5
|
1.3
|
|
Case 2
|
14.2
|
16.9
|
47.1
|
264
|
73.5
|
10.6
|
3.6
|
90.9
|
4.7
|
3.6
|
|
Case 3
|
8.5
|
16.6
|
49.1
|
219
|
95.5
|
20.9
|
1.3
|
101.1
|
5.9
|
3
|
|
Case 4
|
13.3
|
19.9
|
44.3
|
215
|
78
|
10
|
1
|
91.9
|
3.3
|
2.8
|
|
Case 5
|
9.9
|
16.1
|
46.5
|
241
|
102.3
|
11.9
|
2.5
|
78.2
|
3.7
|
4.8
|
|
Case 6
|
16.5
|
16.1
|
45.2
|
237
|
69.4
|
20
|
1.2
|
87.8
|
6.2
|
3.5
|
|
To make a definitive diagnosis, the blood tests were accomplished. The results of the inflammatory parameters showed that that the WBC counts in 4 patients (4/6, 66.7%) and the neutrophil proportions and CRP levels in all patients (6/6, 100%) were higher than normal (Table 1). Acute infection may result in multiorgan functional disturbance; thus, we investigated the parameters of liver function, renal function, myocardial enzymes, arterial blood gas analysis results, coagulation function and so on. The results showed that the patients’ serum prealbumin levels (5/6, 83.3%) were lower than normal, and patients’ PTs (6/6, 100%) and D-dimer (5/6, 83.3%) levels were significantly elevated. Moreover, the patients’ alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin levels, APTTs, fibrinogen levels, renal function parameters (blood urea nitrogen [BUN], creatinine [Cr]), myocardial enzymes (TnI) and arterial blood gas analyses results (pH, PaO2, PaCO2) were normal (Table 1). Interestingly, we also found that the serum iron concentrations in the 6 patients were all lower than normal.
Ultrasonography and intestinal computed tomography (CT) examinations revealed intestinal mucosal edema in 3 patients (3/6, 50%), and ascites in 0 patient.
Routine stool examinations revealed red blood cells (RBCs) and WBCs in the stool of 2 patients (2/6, 33.3%); however, coccus to bacillus (C/B) ratio in all 6 patients could not be analyzed, because the bacterial loads were not sufficient (Table 2). In accordance with the routine fecal test results, fecal occult blood tests (FOBTs) were positive in 2 patients (2/6, 33.3%) (Table 2). The detection of C. difficile toxins and antigens in the stool were negative (Table 2). Stool culture on Salmonella Shigella (SS) agar plates were all positive, the single bacterial colonies were identified with mass spectrometry (MS) and the results indicated Salmonella. Serovar testing revealed all isolated strains were S. enterica serovar Enteritidis (S. Enteritidis) as detected by the Serum agglutination test (Table 2). Taken all, prolonged PT, elevated D-dimer, decreased serum iron and decreased serum prealbumin were associated with the diagnosis of S. Enteritidis infection.
Table 2
The fecal test results of the 6 patients when they were admitted.
|
stool routine test (cells/per high-power field)
|
FOBT
|
C. difficile
|
Stool culture
|
Serovar
|
|
WBC
|
RBC
|
antigen
|
toxin A&B
|
Salmonella
|
S.Enteritidis
|
Case 1
|
8–12
|
10–15
|
+
|
-
|
-
|
+
|
+
|
Case 2
|
0–1
|
0
|
-
|
-
|
-
|
+
|
+
|
Case 3
|
3–5
|
20–30
|
+
|
-
|
-
|
+
|
+
|
Case 4
|
0
|
0
|
-
|
-
|
-
|
+
|
+
|
Case 5
|
0
|
0
|
-
|
-
|
-
|
+
|
+
|
Case 6
|
0
|
0
|
-
|
-
|
-
|
+
|
+
|
The Kinetics Of The Patients'; Symptoms And Clinicopathological Variables
Antimicrobial susceptibility testing was performed using the MIC method with the VITEK 2 COMPACT analyzer. All the isolated Salmonella strains were sensitive to trimethoprim-sulfamethoxazole and levofloxacin and resistant to ampicillin.
Before fecal cultures and antibacterial susceptibility results were obtained, levofloxacin and Bacillus licheniformis capsules were administered according to experience, accompanied by body temperature control, fluid infusion and correction of electrolyte imbalance. According to the antibacterial susceptibility results, we added rifaximin and live combined B. subtilis and Enterococcus faecium enteric-coated capsules to destroy the Salmonella and to modulate the imbalance in the gut microbiota. During the disease course, the body temperatures of 5 patients returned normal on the second day of hospitalization, and the body temperatures of the other 2 patients were controlled on the third day (Fig. 1a). The defecation frequency and stool characteristics (Bristol type) kept decreasing to normal after admission (Fig. 1b). CRP levels increased on the second day and then returned to normal (Fig. 1c); WBC counts (Fig. 1d) and neutrophil percentages (Fig. 1e) continued to decrease after admission. Serum iron levels decreased on the second day and then increased (Fig. 1f). Serum prealbumin levels decreased in the first 3 days and then increased (Fig. 1g). PT increased on the second day and then decreased (Fig. 1h). D-dimer continued to decrease to normal after admission (Fig. 1i). The bowel wall was not detected to be thickening on the 8th day. After hospitalization, 3 additional fecal cultures (on the 3rd, 4th and 5th day respectively) were performed, and the results were all negative. Levofloxacin was withdrawn when a patient’s body temperature remained normal for 3 days and the defecation frequency was less than 3 times per day. Patients were discharged on the 10th day.
Positive associations between decreased serum iron and the severity/outcome of S. Enteritidis infection
In this study, we found that serum iron levels in all patients was lower than normal, and patients with a higher body temperature, higher WBC count and higher neutrophil concentration had lower serum iron level, and with the remission of symptoms and inflammatory parameters, serum iron increased (Table 1 and Fig. 1). We then analyzed correlations between serum iron level and inflammatory parameters and found that the serum iron concentration was negatively associated with body temperature (Pearson correlation coefficient r= -0.458, p = 0.002, Fig. 2a), defecation frequency (Pearson r= -0.524, p < 0.001, Fig. 2b), CRP level (Pearson r= -0.604, p < 0.001, Fig. 2c), WBC count (Pearson r= -0.429, p = 0.005, Fig. 2d), neutrophil proportion (Pearson r= -0.658, p < 0.001, Fig. 2e) and PT (Pearson r= -0.531, p < 0.001, Fig. 2f).
Hepcidin is a cytokine synthesized by liver and plays a critical role in iron metabolism. We then investigated whether hepcidin is involved in iron down-regulation during S. Enteritidis infection. Our results showed that the patients’ serum hepcidin levels (6/6, 100%) were higher than normal when they were admitted, they increased on the second day, and then decreased with symptom remission (Fig. 3a). Serum hepcidin was negatively correlated with serum iron (Pearson r= -0.889, p < 0.001, Fig. 3b). Above all, our results indicated that the decreased serum iron was associated with the severity and outcome of S. Enteritidis infection, and that the elevated serum hepcidin was associated with the decreased serum iron during S. Enteritidis infection.
Positive associations between the prolonged PT/elevated D-dimer and the severity /outcome of the S. Enteritidis infection
We then investigate the correlations between the coagulation system disturbance and the severity and outcome of S. Enteritidis infection. The patient with severe symptoms had a higher D-dimer and longer PT than the other patients, and with the remission of symptoms and inflammatory parameters, PTs and D-dimer levels were decreased (Table 1 and Fig. 1). PT was positively associated with body temperature (Pearson r = 0.447, p = 0.003, Fig. 4a), defection frequency (Pearson r = 0.566, p < 0.001, Fig. 4b), CRP level (Pearson r = 0.69, p < 0.001, Fig. 4c), WBC count (Pearson r = 0.544, p < 0.001, Fig. 4d) and neutrophil proportion (Pearson r = 0.612, p < 0.001, Fig. 4e); serum D-Dimer concentration was positively associated with body temperature (Pearson r = 0.823, p < 0.001, Fig. 4f), defection frequency (Pearson r = 0.693, p < 0.001, Fig. 4g), CRP level (Pearson r = 0.512, p = 0.001, Fig. 4h), WBC count (Pearson r = 0.752, p < 0.001, Fig. 4i) and neutrophil proportion (Pearson r = 0.884, p < 0.001, Fig. 4j). Taken together, the prolonged PT and elevated D-Dimer were correlated with the severity and outcome of the S. Enteritidis infection.
The Correlation Between Serum Prealbumin And Acute Inflammation Status
Serum prealbumin and albumin are indicators of liver synthesis function, and prealbumin may decrease earlier than albumin when liver function is damaged. As shown above, the decreased prealbumin was associated the diagnosis of S. Enteritidis infection. We then investigated the association between serum prealbumin and the severity and outcome of S. Enteritidis infection, and found that it was negatively correlated with CRP level (Pearson r= -0.678, p < 0.001, Fig. 5a) and PT (Pearson r= -0.353, p = 0.022, Fig. 5b); there was no significant correlations between serum prealbumin and body temperature (Pearson r = 0.208, p = 0.187, Fig. 5c), defection frequency (Pearson r= -0.138, p = 0.383, Fig. 5d), WBC count (Pearson r= -0.073, p = 0.646, Fig. 5e) and neutrophil proportion (Pearson r= -0.093, p = 0.559, Fig. 5f).
As the serum prealbumin continued to decrease in the first 3 days, and begun to increase at the 4th day, while other parameters of inflammation begun to restore at the 2th or 3th day. We then analyzed the correlation between serum prealbumin and inflammatory parameters of the first day, and found that although the correlation coefficients were larger than 0.2 or smaller than − 0.2, the p values were all larger than 0.05 (Fig. 5g-5 l), this could be reasoned by the small case number. Taken together, our results indicated that a decreased prealbumin was correlated with the diagnosis of the S. Enteritidis infection, but more solid proofs were needed to verify its association with the severity and outcome of S. Enteritidis infection.
The kinetics of serum homocysteine during S. Enteritidis infection
When the patients were admitted, all patients had symptoms of diarrhea, thus, a low-fat and low-protein diet was provided. A normal diet was reintroduced when a patient’ defecation frequency was less than 3 times per day and a patient’ fecal Bristol type returned to type 5. However, during the disease course in these 6 patients, we found that the patients’ homocysteine levels continued to increase beginning on the 6th day of hospitalization, and 2 patients’ homocysteine levels were higher than normal on the 7th day (Fig. 6a). The patients were aged from 18 to 23 years, their blood pressures were normal, and none had a family history of high blood pressure; therefore, other mechanisms responsible for the increased serum homocysteine may exist. Serum folate plays a critical role in homocysteine metabolism. Accompanied by methyltetrahydrofolic acid, homocysteine can be transformed into methionine and tetrahydrofolic acid by methylenetetrahydrofolate (MTHFR). We found that the patients’ serum folate levels were either lower than normal or at the lower normal limit (7 nmol/L ~ 45.6 nmol/L) on the 8th day. We provided folate tablets (5 mg, QD) to the patients with high serum homocysteine and asked the other patients to improve their nutrition and consume more green vegetables for folic acid supplementation. Then, serum folate increased (Fig. 6b), while serum homocysteine decreased (Fig. 6a). Thus, we suggest that a normal diet should be reintroduced early.