Recently, immunosuppression is thought to be due to increased immunoregulatory activity through activation of Treg cells⁸. In ischemic stroke CD4+CD25 T-Regulatory cells play a key part in controlling the immune mechanism. A rapid loss and functional deactivation of T cells are common changes in stroke patients consistent with immunodepression after brain ischemia. After severe brain injury, CD4 cells play a role in the down regulation of innate immune responses that play an important role in suppressing the immune response, in maintaining immunologic homeostasis and preventing autoimmunity and this is likely to also be the case after stroke⁹. A stronger decrease in cellular immune responses and an increased sympathetic activity after stroke are associated with a higher risk of infections¹⁰. The current data suggests that prediction or early diagnosis of post stroke infection was not yet achieved as there were no specific bedside laboratory markers of impending infection after stroke. The predictive value of these parameters could provide an early and easily obtainable parameter to identify patients at high risk of subsequent infection¹¹. 

We aimed to ascertain the frequency of post stroke infections and to elucidate which stroke-specific clinical factors and immunological alterations is the key characteristics that would allow the prediction of those patients who are at highest risk for post stroke infections.

 

PATIENTS AND METHODS

 

Study Population             

Twenty five ischemic stroke patients in the first 48 hours of stroke onset who were admitted to the Neurology Department, Fayoum University Hospital, were enrolled. Twenty age and sex matched healthy subjects were chosen as a control group. The study was approved by the local ethical committee of the Faculty of Medicine, Fayoum University, and all the participants or their close relative consented to this study. Patients with TIA, intracerebral or subdural hemorrhage, or those presented after the first 2 day of the stroke onset or patients below age of 40 years old, or those with history or documentation of infection within 2 weeks prior to the stroke onset, or patients had autoimmune disease or those received immunosuppressant or antibiotics within the preceding 3 months were excluded from the study.

All patients underwent full history taking with special emphasis on stroke risk factors and history suggestive of infection. Thorough general and neurological examination; with special notation on the vascular examination; were done. All patients were assessed on admission to the hospital, followed up daily for 1 week then examined again after1 week. Stroke severity was assessed by National Institutes of Health Stroke Scale [NIHSS] and The Barthel's Index (BI) was used to assess the activities of daily living both on admission and at follow up. The presence of chest infection was determined according to Mann criteria¹². Specifically, patients were required to have 3 or more of the following characteristics: fever (38°C), productive cough with purulent sputum, abnormal respiratory examination (tachypnea 22/ min, tachycardia, inspiratory crackle, bronchial breathing), abnormal chest radiographic findings; pulmonary infiltration; arterial hypoxemia (PO₂ 70 mmHg or SpO₂ 94%), and isolation of a relevant pathogen (positive Gram’s stain and culture). Urinary tract infection was considered if the patient had dysuria or urgency, or presence of pus cells in urine analysis.

Chest X ray was done day 1 and 7 to look for evidence of chest infection.

Computerized Tomography (CT) or MRI Brain was performed for all patients on admission for the diagnosis of ischemic brain infarction and exclusion of intracerebral and subarachnoid hemorrhage.

 

Flow cytometric analysis of T regulatory cells:

The percentage of Treg cells was estimated by flow cytometric (FCM) analysis of T lymphocytes expressing CD4 and CD25 as described by Urra et al.¹³. Following incubation, cells were washed twice with PBS. Analysis of T regulatory (Treg) cells was done using EPICS ELITE Coulter FCM. Lymphoid cells were gated according to their forward and side light scatter properties. The percentage of the mononuclear cells expressing CD4 and CD25 within the gated population represents the percentage of Treg cells.

 

Statistical Analysis

The data were analyzed using the SPSS software version 15. Chi-Square Tests used to compare between clinical data in patients’ subgroup. Fisher's Exact Test was used for comparing dysphagia between patients’ subgroup. Sample independent test were used to compare NIHSS, Barthel's index and laboratory parameters means between patients’ subgroup. P ≤ 0.05 was considered statistically significant.           

 

RESULTS

 

The patients’ age ranged from 51 to 78 years with mean age of 62.56±6.801. They were 15 men and 10 women. Stroke was on the right side in 19 patients whereas 6 had left side stroke. Six patients were hypertensive, five were diabetic, one patient was hypertensive and diabetic and 9 patients had heart disease. The National Institutes of Health Stroke Scale [NIHSS] mean was 11.56±3.367 on admission and 10.72±0.980 at follow up. The Barthel's Index (BI) mean was 12.80±11.644 on admission and 22.00±11.180 at follow.

We demonstrate a significantly higher CD4/ CD25 mean in stroke patients both on admission and at follow up in comparison to the control group (Table 1). The control group was age and sex matched (P, 0.6 & 0.9 respectively).

CD4+cells in stroke patients were lower than that of the control group, CD25+ cells were higher in stoke patients both on admission and at follow up yet the difference did not reach statistically significant level.

We found the incidence of post stroke infection (28%). Of our patients (24%) developed chest infection and (4%) had got urinary tract infection (UTI) (Figure 1).

 

Clinical data in patients' subgroups

Comparing Group (2); Patients with post stroke infection with Group (1) Patients; without infection revealed that the mean NIHSS on admission and at follow up, the presence of dysphagia and positive chest X ray were statistically significantly higher in Group (2); Patients with post stroke infection. Group (2) patients had older age and lower Barthel's index on admission and at follow up in comparison to Group (1) yet this did not reach statistical significance. Other clinical data and risk factors did not differ between the patients' subgroups (Table 2).

 

Laboratory parameters in patients' subgroups:

No significant differences were encountered between patients' subgroups in T cell subset whether on admission or at follow up (Table 3).

No significant differences were found in the frequency of T regulatory cells on admission and at follow up after 1 week in stroke patients with infection (p=0.224).

No significant correlations were encountered between T helper cells CD4+, CD25+ or CD4+/CD25+ ratio and functional outcome  as measured by Barthel index in stoke patients as a whole or in patients subgroup with or without infection.

 

Table 1. T-regulatory cells subsets in the patients in comparison to the control.

 

T-regulatory cells subsets		Stroke patients (n = 25)	Control group (n = 20)	P value
On admission	CD4	36.42±11.26	44.05±4.6	0.166
	CD25	5.15±6.1	1.88±0.9	0.281
	CD4+CD25+	8.63±4.91	2.1±0.978	0.032*
At day 7	CD4	37.91±9.5	44.05±4.6	0.178
	CD25+	10.5±6.9	1.88±0.9	0.752
	CD4+CD25+	8.266±4.26	2.1±0.978	0.041*

* Significant at p<0.05

 

 

 

Figure 1. Incidence of post stroke infection.

 

Table 2. Clinical data in patients' subgroups.

 

Clinical Parameters	Group (1) Patients without infection (18/25- 72%)	Group (2) Patients with post stroke  infection (7/25 - 28%)	P-value
Age (Mean±SD)	61.5±6.8	66.1±3.4	0.114
Sex (Male: female)	12:6	3:4	0.295
Presence of dysphagia	0	5	0.05*
Dysuria	1	1	0.49
Pus cell in urine analysis	1	1	0.49
Urinary catheter	4	2	0.98
Atrial fibrillation	3	3	0.44
Ischemic heart disease	4	2
Presence of diabetes Mellitus	3	2	0.78
The mean NIHSS at admission	10.6±3.2	14±2.64	0.012*
The Barthel's Index at admission	15.3±11.56	6.42±9.88	0.088
Positive chest X ray	4	1	0.004*
The NIHSS at follow up	10.4±0.92	11.4±0.78	0.027*
The Barthel's Index at follow up	24.44±11.1	15.7±9.32	0.079

NIHSS National Institutes of Health Stroke Scale                    

* Significant at p<0.05

Table 3. Laboratory parameters in patients' subgroups.

 

Laboratory parameters		Group (1) Stroke patients without infection (n=17)	Group (2) Stroke patients with infection (n=7)	P-value
T-cell subsets on admission	CD4+	38.32±11.66	31.82±9.41	0.224
	CD25+	5.96±8.31	7.54±7.55	0.670
	CD4+CD25+	9.14 ± 5.21	7.40 ± 4.21	0.442
T-cell subsets at follow up	CD4+	38.45±7.78	36.6 ± 13.66	0.675
	CD25+	10.01-7.1	11.67 ± 6.9	0.607
	CD4+CD25+	8.01± 4.4	8.87 ± 4.18	0.666

 

 

DISCUSSION

 

The third most common stroke complication is infection¹⁴. Post stroke infectious is increasingly recognized as the leading cause of death in the post-acute phase of stroke³. This infection occurs within the context of complex interplay between the brain and immune systems⁷. Considerable debate continues as to the extent to which the post stroke infection occurred. Infectious complications, predominantly chest and urinary tract infections, have been variably reported to occur in 23-65% of all stroke patients within the first few days after stroke¹. This high incidence of infections is likely to be a result of an impaired immune function¹⁵. Thus we thought to explore the prevalence, clinical and immunosuppression contribution to post stroke infection in a hospital based study.

We found; in accordance to Tanzi et al.¹⁶ the incidence of post stroke infection (28%). But higher than Vermeij et al. study¹¹, who reported stroke-associated infection in 15% of their patients.

Previous studies reported wide variation in the incidence of UTI, between 3% and 44% of acute stroke patients^{17-19. In this study in accordance to} Aslanyan et al.¹⁴, we found the incidence of urinary tract infection was; only 4%; lower than those reported by other studies^18,19 and this difference could be attributed to the difference in the length of follow-up. The median time to developing UTI as reported by Davenport et al.²⁰ is around 15 days. One of the limitations in our study was short follow up period for only one week; during their stay in the hospital and this may explain the encountered lower incidence in the current study.

The mechanisms leading to an increased susceptibility to infections after stroke are still poorly understood⁴. Several studies have attributed the increased infection vulnerability of patients with acute ischemic stroke to stroke-induced immunosuppression¹⁵. Previous studies documented T cell activation after stroke could be a non-specific response or a peculiar response directed against specific brain antigens. It was found that an increase in the percentage of CD4+^.Tregs which comprise about 10% of the CD4+ T cell population²¹. Natural Treg cells expressing CD4 and CD25 and participate in the immune response to perhaps all infectious agents. They have a role in defending against infections and in maintaining immunological self-tolerance²². There is still a dilemma whether the regulatory T cells (Treg) favorably affect the outcome by limiting inflammatory response in and around the infarcted area or can be harmful to the host through clinical immunodepression and increase susceptibility to infection²³. Some studies reported evidence of immune activation and Treg cells in acute ischemic stroke, and potentiation of cerebroprotective role of Treg cells as major modulators of post ischemic/inflammatory brain damage²⁴. Oppositely, other study observed that, there was marked decline of Tregs after stroke¹³.

In this study, in accordance to Urra et al.¹³, we demonstrate a significantly higher CD4/CD25 ratio in stroke patients both on admission; P: 0.032; and at follow up; P: 0.041; in comparison to the control group. In stroke patients with infection, no significant difference was found between the frequency of T regulatory cells on admission or at after 1 week (P; 0.224). In the current study, there was no statistical difference in the percentage of Treg cells between patients who had post stoke infection and those who did not neither at presentation nor at follow up. CD4+cells in in stroke patients on admission are lower than that of the control group, and CD4+cells in in stroke patients with infection at follow up are lower than that of those without infection, yet the difference did not reach statistically significant level. Previous study reported more pronounced and persistent loss of CD4T cells from peripheral blood in stroke patients who developed infections than patients without infections due to a lack of recovery in the infected patients²⁵. The absence of significant difference in our study could be contributed to the small number of our patients. This is post stroke immunosuppression may result from a state of stress-mediated reduced immune competence²⁶. The complex series of alterations in peripheral immune pathways after stroke occurred via stimulation of the hypothalamo pituitary-adrenal axis and the sympathetic nervous system⁵. Activation of the sympathetic nervous system is the main immunosuppressive mechanism that predisposes stroke patients to life-threatening infections¹⁵. An excessive activation of inhibitory neuroendocrine pathways without systemic inflammation can inappropriately suppress the immune system and increase the risk of infections¹⁰.

In summary, Treg cells in this study did not show a relevant association with the development of infections. Understanding the unique properties of natural Treg cells and their mode of action may result in new therapeutic avenues useful for the control of infectious diseases. In accordance to other studies ^{[12, 27]} we identified dysphagia and stroke severity as predictors of infection after stroke. We did not find   association of post stroke infection with other clinical or laboratory parameters.

 

Limitations of the study

Our study does have a number of limitations. First and mostly the number of the patients were small. Unfortunately the study was not financially supported and the researcher himself paid for the cost of the study. The follow up period was short only for one week during the patients stay in the hospital. Samples were taken within 48 hours. Some studies show immune alteration as early as 12 hours. 

 

Conclusions and Recommendations

In this study post stoke infection was found to be frequent. Stroke patients with dysphagia and those with severe stroke are more liable to develop post stroke infection. Further studies with larger number of patients, longer follow up period and enrollment of patients earlier within first 12 hours of onset are needed to validate this finding and to find significant differences.

 

[Disclosure: Authors report no conflict of interest]

 

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