| ObjectiveEnterovirus 71 (EV71) infection can cause severe central nervous system (CNS) damages, such as encephalitis, brainstem encephalitis, acute flaccid paralysis and so on, and some patients may develop cardiopulmonary failure (CPF), resulting in high mortality and morbidity. Their pathogenesis is unclear, but may be associated with over-activation of the sympathicus as a result of damaged CNS, followed by neurogenic pulmonary edema (NPE) and cardiovascular system function changes, eventually leading to CPF. Considering that there are no effective antiviral drugs against EV71 infection, EV71 infection is mainly treated by implementing staged management and comprehensive symptomatic therapy. Therefore, the key to reducing the mortality of EV71 infection and the incidence of its sequelae is to investigate typical clinical manifestations and/or laboratory indices so as to detect potential CPF, against which correct treatment regimes are taken to block the development of the disease. Recent studies found that cell-encoding miRNA molecules are closely related to EV71 infection and replication. MiR-206 molecule and its target gene CCL2 influence EV71 infection, and this can be verified by bioinformatic analysis, but few studies are available in this field worldwide on whether MiR-206 molecule and its target gene CCL2 are associated with the severity of the disease. Our study aims to evaluate the correlation of the early clinical manifestations and laboratory examinations of CPF with the severity of CPF, and discuss the risk indicators for CPF by investigating the typical clinical manifestations and supplementary laboratory indices in children suffered with V71 infection and accompanying CNS damages, in conjunction with the expression levels of miR-206 molecule and its target gene CCL2 in cerebrospinal fluid. High-dose steroid pulse therapy for severe EV71 infection remains controversial, for it is impossible to explain the validity of this therapy in terms of the possible pathogenesis of CPF, and this therapy also has severe side effects. To determine the clinical value of glucocorticoid in treating severe EV71 infection, in our clinical studies, pediatric patients with EV71 encephalitis were randomly divided into a steroid pulse therapy group and a non-steroid pulse therapy group and their clinical data including efficacy and side effects were compared using statistic analysis.MethodsHMFD pediatric patients with EV71 encephalitis who had received treatment at the Taian Central Hospital from February 2014 to December 2014 were included in this study. The patients were divided into two groups according to whether they had CPF:the severe EV71 encephalitis group included EV71 HFMD patients with concurrent CNS damage and CPF symptoms; the mild EV71 encephalitis group included patients with CNS damage but without CPF. Firstly, the two groups were. compared in terms of age, gender, body weight, duration of fever, length of stay, vital signs, laboratory examinations and prognosis, with the performance of neurological, respiratory and circulatory systems as well as the differences in the results of laboratory examinations being highlighted. The risk factors for progression of severe EV71 infections into CPF were determined by one-way and multi-way analysis of variance. Secondly, RNA isolation and real-time quantitative fluorescent PCR were used to determine the expression levels of miR-206 and CCL2 mRNA in all cerebrospinal fluid (CSF) specimens from the two groups, the Student's t-Test was used to compare the difference in expression between the groups, and Pearson rank correlation analysis was used to evaluate the correlation between the expression levels of miR-206 and CCL2. Thus, their roles in the development of EV71 encephalitis into CPF may be determined. Finally,80 pediatric patients with EV71 encephalitis were randomly divided into a steroid pulse therapy group and a non-steroid pulse therapy group according to whether high-dose methylprednisolone pulse therapy was used. The Student's t-Test was used to analyze the clinical data of the two groups, such as duration of fever, length of stay, vital signs, laboratory examinations and prognosis before and after the treatment in the children. Based on the differences in the clinical data, and the efficacy and side effects between the two different treatment options, as well as their influences on diagnosis, we may identify the advantages and disadvantages of high dose methylprednisolone in the treatment of EV71 encephalitis.Results1. Risk factors for cardiopulmonary failure after EV71 infection.173 HMFD pediatric children with EV71 encephalitis were included in this study. There were 61 (35.3%) children in the severe EV71 encephalitis group, including 42 males,19 females, aged (23.70±12.93) months; 112 (64.7%) children in the mild group, including 74 males and 38 females, aged (34.17+20.24) months. There were statistically significant differences in age and weight between the two groups (P< 0.05); the differences between the two groups in gender ratio and the degree and duration of fever were not statistically significant (P= 0.710,0.066 and 0.089 respectively). Moreover, the nervous system involvement duration and length of stay in the severe group were significantly longer than those in the mild group (P <0.05). The neurological system symptoms include:headache (27.7%), vomiting (46.8%), lethargy (34.7%), irritation (22.5%), abnormal eye movement (13.3%), limb weakness (22.0%), sweating (26.6%), myoclonus (30.6%), cervical rigidity (26.0%), knee reflex hyperactivity (28.9%), knee reflex hypoactivity (11.0%), hypermyotonia (20.2%), positive Babinski sign (28.3%), ataxia (19.1%), limb tremor (12.7%) and seizures (6.4%). According to one-way analysis of variance, as compared with the mild group, the severe group had significantly higher incidences of vomiting, lethargy, irritation, abnormal eye movement, limb weakness, myoclonus, knee reflex hypoactivity, ataxia, and limb tremor (P< 0.05). Similarly, the incidences of tachycardia, tachypnea, extended CRT, hepatomegaly, moist rales, and exudative X-ray finding in the severe group were significantly higher than those in the mild group (P< 0.05). On the other hand, the difference between the two groups in blood pressure was not statistically significant. The laboratory examinations of all the children are as follows:white blood cell count (WBC,11.50 ±4.45×10?9/L), hemoglobin (HB,117.46±11.36 g/L), C-reactive protein (CRP, 8.29±8.18 mg/L), platelet count (PLT,220.51±81.24×10?9/L), blood glucose (GLU,7.34±2.32 mmol/L), alanine aminotransferase (ALT,32.12±22.50 U/L), aspartate aminotransferase (AST,44.66±29.64 U/L), creatine kinase musclebrain isoenzyme (CK-MB,36.17±20.99 U/L), creatine kinase (CK,194.79±110.19 U/L), lactate dehydrogenase (LDH,305.35±178.11 U/L), K (3.85±0.33 mmol/L), Na (136.27±5.95 mmol/L), and Cl (102.10±6.37 mmol/L). One-way analysis of variance found that the levels of blood glucose and white blood cell in the severe group were significantly higher than those in the mild group (P< 0.05), but there were no significant differences between the two groups in hemoglobin, CRP, platelet count, ALT, AST, CK-MB, CK, LDH, K, Na and Cl. Multivariate logistic regression analysis showed that leukocytosis, hyperglycemia, vomiting, lethargy, irritation, abnormal eye movement, limb weakness, myoclonus and limb tremor were the high risk factors for CPF after HFMD infection.2. Downregulation of CCL2 as a result of upregulation of microRNA-206 is correlated with the cardiopulmonary failure after EV71 infection.In order to determine whether miR-206 inhibits endogenous EV71, we used qRT-PCR to measure the expression levels of miR-206 and CCL2 in cerebrospinal fluid. A total of 74 patients, including 40 patients with mild EV71 encephalitis and 34 patients with severe EV71 encephalitis were studied. The expression levels of miR-206 showed significant difference between the severe group and the mild group (3.10±1.95:3.95±2.61, t=9.275, P< 0.001), suggesting that when severe EV71 encephalitis developed into CPF, the expression levels of miR-206 were downregulated. The levels of CCL2 in the severe group were significantly higher than those in the mild group (4.53±3.18:3.01±2.16, t=8.309, P< 0.001). The results showed that the expression level of CCL2 was up-regulated in the progression of EV71 encephalitis into CPF. The correlation between the miR-206 and CCL2 expression values was evaluated using Pearson rank correlation analysis. The results indicated a statistically significant negative correlation (R=-0.5202, P <0.05).3. Efficacy of high dose methylprednisolone pulse therapy in the treatment of EV71 infection.The difference between the methylprednisolone treatment group and the non-steroid treatment group in nervous system involvement, staging and clinical data was not statistically significant (P> 0.05). Similarly, vital signs and laboratory examinations were not significantly different when the two groups of children were admitted to hospital.48-72 h after hospitalization, the difference in SBP, DBP, CRP, Hb and PLT between the two groups was not statistically different. On the other hand, the HR (144.13 ± 15.24:137.43± 10.75, t= 2.950, P= 0.005), RR(38.18 ± 8.05:35.20 ± 6.04, t= 2.423, P= 0.020), WBC (14.87 ± 3.72:12.75 ± 3.32, t= 2.896, P= 0.006) and GLU (9.09 ± 3.87:7.74 ± 2.28, t= 2.067, P= 0.045) in the steroid therapy group were significantly higher than those in the non-steroid therapy group (P< 0.05).5 children had sequelae at the time of discharge and were considered improved, including 1 case with flaccid paralysis and 1 case with epilepsy in the steroid treatment group; and 1 case with flaccid paralysis,1 case with swallowing dysfunction, and 1 case with cognitive function obstacles in the non-steroid treatment group.2 patients in the steroid pulse therapy group died. The difference in cure rate between the two groups was not statistically significant (P> 0.05).Conclusion1. According to the clinical manifestations and related risk factors analysis of EV71 infection children in our study, the high risk factors for CPF after HFMD infection include many neurological manifestations, such as leukocytosis, hyperglycemia, vomiting, lethargy, irritation, abnormal eye movement, limb weakness, myoclonus and limb tremor.2. In the study, CCL2 was validated as a direct target of miR-206, and upregulation of CCL2 caused by downregulation of miR-206 is responsible for the development of severe EV71 infection. It may be used as a risk factor for CPF after EV71 infection.3. High dose methylprednisolone pulse therapy for treating EV71 infection can't shorten the course or improve the prognosis of the disease. In contrast, it has side effects and might aggravate the disease or interfere with its diagnosis. Our study suggested that there is no beneficial effect to use high-dose steroid pulse therapy for the treatment of EV71 infection.In conclusion, our study focused on severe EV71 infection and the difficulties in its clinical research. Our results are instructive for clinical practices. In case of EV71 infection, its mortality and the incidence of its sequelae can be reduced by detecting the aforesaid risk factors early, taking preventive measures timely, avoiding application of glucocorticoid therapy and determining the correct treatment regime. |
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