Rapid Diagnosis Of Herpes Encephalitis In Children By PCR-microarray Technology For Simultaneous Detection Of Seven Human Herpes Viruses

The viral encephalitis is one of inflammations in the menings and brain tissue by the viruses. It is the most familiar cause of the infectious diseases in the central nervous system, and that the herpes viruses are common pathogens in the viral encephalitis. Among the herpes viruses, herpes simplex virus type 1, type 2(HSV-1,HSV-2), varicella-zoster virus(VZV), Epstein-Barr virus(EBV), cytomegalovirus(CMV) and human herpes virus 6(HHV-6A, HHV-6B) are representative. The herpes encephalitis has a higher sick rate and mortality rate. It is easy to induce the serious functional defect without treatment in time. The earlier antiviral therapy can reduce the mortality remarkably and enhance livability, so the early diagnosis is very important in herpes encephalitis, while the early diagnosis and therapy in time is dependent on the laboratory diagnosis. In this study, the multiple primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses, and the multiplex PCR-microarry technology was established which could simultaneously detect herpes simplex virus type 1,type 2(HSV-1,HSV-2), varicella-zoster virus(VZV),Epstein-Barr virus(EBV) , cytomegalovirus(CMV) and human herpes virus 6(HHV-6A,HHV-6B); A total of 290 cerebrospinal fluid (CSF) specimens from children with suspected viral encephalitis were detected by PCR-microarray. The results indicated that this multiplex PCR-microarray technology proved to be an effective way for detecting and species identification of seven human herpes viruses.ObjectiveTo establish a new technique which can rapidly detect common human herpes viruses at the same time and to explore a new method for rapid diagnosis of herpes encephalitis in children.MethodsThe multiplex primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses, and the PCR- microarry technology was established which could simultaneously detect herpes simplex virus type 1, type 2(HSV-1, HSV-2), varicella-zoster virus(VZV), Epstein-Barr virus(EBV), cytomegalovirus(CMV), and human herpes virus 6(HHV-6A, HHV-6B); A multiplex PCR-microarray technology for simultaneous detection and species identification of seven human herpes viruses was to be established. A total of 290 cerebrospinal fluid (CSF) specimens from children with suspected viral encephalitis were detected by PCR-microarray technology. The results were compared with those of TaqMan PCR. The specimens positive for human herpes virus DNA were further confirmed by sequencing after cloning. In addition, 30 children without viral infection were enrolled as the negative controls (eighteen with falling sickness and twelve with purulent meningitis).ResultsThe human herpes PCR-microarray technology can detect more than more than 10 copy viral magnitude. The every sign of probes was not disturbed each other in herpes viruses. There were no cross-reaction with bacteria, epiphyte, hepatitis B virus (HBV) and human DNA. Among 290 cerebrospinal fluid specimens from children with suspected viral encephalitis, eleven (11/290, 3.80%) were tested positive by PCR-microarry. Among them , HSV-1 was 3, HSV-2 was 2, EBV was 1, CMV was 1,HHV-6B was 1, mixed infection(HSV-2/CMV) was 1. Compared with those of TaqMan PCR, the sensitivity and specificity of the PCR-microarray technology was 97.1% and 100%; Eleven positive specimens were sequenced after cloning, and the results of sequencing were aligned with the sequences of standard virus strains. The homology was higher than 98%. None of the 30 control CSF was positive for human herpes virus DNA by the PCR-microarray technology. We made a search for 11 cases which were positive, and found two cases were herpes encephalitis which were definitely diagnosed in clinic, while another nice cases were viral encephalitis which were acatalepsia. The PCR-microarray technology provides an effective way to identify the cause of diseases and guide clinical antiviral selection.ConclusionsThis multiplex PCR-microarray technology, which could simultaneously detect and identify seven human herpes viruses, is rapid, specific and sensitive. It will provide a early, specific and etiologic diagnosis in children with herpes encephalitis.