Twenty-six patients with RE were enrolled in this study, including 16 girls (61.5%) and 10 boys (38.5%). Their mean ages were 5.8 years (range: 1.2-18 years) and 7.5 years (range: 2.4-18.5 years) at seizure onset and at time of surgery, respectively. History of infection and/or viral vaccination prior to the seizure onset was observed in 14/26(53.8%) patients. Eleven patients (42.3%) underwent left-side surgery, including functional hemispherectomy (FH), anatomic hemispherectomy (AH), and hemisphere disconnection. The etiologies for the control group consisted of ganglioglioma (n=5), hippocampal sclerosis (n=8), and focal cortical dysplasia (n=7). The baseline comparison between RE and control groups is shown in Table 1.
RE (n = 26) Non-RE (n = 20) Gender (female) 10 (38.5%) 12 (60%) Preceding infection 14 (53.8%) 3 (6%) Mean age at seizure onset (years) 5.8 8.6 Mean age at surgery (years) 7.5 14.8 Side of resection (left) 11(42.3%) 13 (65%) Surgical method FH 15 (46.2%) 0 AH 2 (15.4%) 0 Disconnection 9 (26.9%) 0 Lobectomy 0 20 (100%) Note: FH: functional hemispherectomy; AH: anatomic hemispherectomy
Table 1. Clinical characteristics of patients with and without RE
Mild to severe progressive unilateral multifocal cortical atrophy around the sylvian fissure and diffuse subcorti-cal hypersignal confined within the involved hemisphere in the FLAIR sequence were observed in all patients (Figure 1A, 1B). The pathological changes, including pyknosis, loss of neurons, diffuse lymphocyte infiltration, perivascular lymphocyte cuffing, and glial cell prolif-eration (Figure 1C, 1D), were observed in RE brain tissues. However, no obvious morphological features of viral inclusion were observed.
Figure 1. MRI and pathological changes in the brains of patients with RE. Atrophy of the right hemisphere cortex and widening of the sulcus (arrowheads) and cau-date nucleus (arrow) were noted before operation (A). Diffuse hypersignal in the subcortical area (arrows, B). Microglial nodule formation in the cortex (arrow, C). Lymphocyte infiltration and features of perivascular cuff (arrow, D).
Among the 26 patients with RE, 23(88.5%) showed positive immunostaining for the HCMV pp65 protein. Among these 23 cases, nine (34.6%) were strongly positive, nine were moderately positive, and five (19.2%) were weakly positive (Figure 2, Table 2). Similarly, HCMV pp65 DNA was detected in 69.2%(18/26) of RE samples; among them, eight (30.8%), six (23.1%), and four (15.4%) were strongly, moderately, and weakly positive, respectively (Figure 3, Table 2). HCMV pp65 protein was predominantly expressed in the cytoplasm of neuron-like cells (Figure 2). In the control group, only two cases (2/20, 10%) were weakly positive for pp65 antigen, and all cases were negative for pp65 DNA (Figure 2, Table 2; P < 0.05 for both). Notably, all of the 18 HCMV pp65 DNA-positive RE samples were positive for HCMV pp65 protein (Table 3). In other words, five IHC-posi-tive patients with RE showed negative ISH staining, in-dicating that combined application of the two methods may be necessary for enhancement of the detection rate.
Figure 2. Expression of HCMV pp65 protein in patients with RE. The pp65 protein was detected by IHC. Representa-tive images are shown at low (200×, upper panels) or high (400×, lower panels) magnification. Positive staining of pp65 was predominantly observed in the cytoplasm of cells (arrows).
IHC ISH RE (n = 26) Non-RE (n = 20) RE (n = 26) Non-RE (n = 20) Positive 23 (88.5%) 2 (10%) 18 (69.2%) 0 Strong 9 (34.6%) 0 8 (30.8%) 0 Moderate 9 (34.6%) 0 6 (23.1%) 0 Weak 5 (19.2%) 2 (10%) 4 (15.4%) 0 Negative 3 (11.5%) 18 (90%) 8 (30.8%) 20 (100%)
Table 2. Summary of IHC and ISH data for brain tissues of patients with and without RE
Figure 3. Expression of HCMV pp65 DNA in patients with RE. HCMV pp65 DNA was detected by ISH. Representative images are shown at low (200×, upper panels) or high (400×, lower panels) magnification. Positive staining of pp65 DNA was predominantly observed in the cytoplasm and nucleus of cells (arrows).
Cases IHC (positive) IHC (negative) Total ISH (positive) 18 0 18 ISH (negative) 5 3 8 Total 23 3 26
Table 3. Comparison of IHC versus ISH results in this study
Based on the IHC intensity of HCMV pp65 antigen, patients with RE were divided into two groups: group A with negative or weakly positive pp65 staining, and group B with moderately or strongly positive pp65 staining. As shown in Figure 4, patients in group B showed a shorter mean duration of the prodromal stage (17.9 ± 5.1 versus 4.6 ± 0.56 months, respectively; Figure 4A; P < 0.05) and a younger age at seizure onset as compared with group A patients (4.5 ± 0.5 versus 7.4 ± 0.9 years, respectively; Figure 4A; P < 0.01). Additionally, patients with RE with more intense HCMV pp65 staining showed a more severe hemisphere cortical atrophy (Figure 4B; P=0.037). There were no correlations between the HCMV pp65 staining intensity and other clinical parameters.
Figure 4. Association between HCMV pp65 antigen expression and the clinical parameters of patients with RE. Patients with RE were divided into group A (nega-tive or weakly positive for pp65 staining) and group B (strongly and moderately positive for pp65 staining). Associations of prodromal stage duration, age at seizure onset (A), and severe atrophy grade (B) with pp65 staining. *Atrophy grade (AG) was defined by the following principles: no (0 point), no enlargement of lateral ventricle and hemispheric sulcus; mild (1 point), widening of the hemispheric sulci without obvious enlargement of lateral ventricles; moderate (2 points), obvious widening of the hemispheric sulci with mild to moderate enlargement of lateral ventricles (less than double wide than contralateral in axial view); severe (3 points), obvious widening of the hemispheric sulci with severe enlargement of lateral ventricles (more than twice as wide as the contralateral axial view).