ABSTRACT
Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml(-1) NS1. Up to 1µgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.
Subject(s)
Antigens, Viral/analysis , Clinical Laboratory Techniques/methods , Encephalitis, Japanese/diagnosis , Viral Nonstructural Proteins/blood , Viral Nonstructural Proteins/cerebrospinal fluid , Virology/methods , Agouti Signaling Protein , Animals , Antibodies, Monoclonal , Antibodies, Viral , Blood Chemical Analysis , Calcium-Binding Proteins , Cell Line , Cerebrospinal Fluid/chemistry , Cyclic GMP-Dependent Protein Kinases , Drosophila , Drosophila Proteins , Female , Flavivirus Infections , GTP-Binding Proteins , Humans , Immunoassay/methods , Immunoglobulin G , Membrane Proteins , Mice , Mice, Inbred BALB C , Mice, Inbred C3HABSTRACT
The involvement of the central nervous system in dengue infections has been reported in countries where the disease in endemic. The purpose of this study was to determine whether an enzyme-linked immunosorbent assay kit designed to detect the dengue NS1 antigen in serum was able to detect this antigen in cerebral spinal fluid (CSF) samples from patients with fatal outcomes. To evaluate the sensitivity of the kit, 26 dengue-positive CSF samples were used. The Pan-E Dengue Early kit was able to detect the NS1 antigen in 13 of 26 dengue-positive CSF samples, resulting in a sensitivity of 50% (95% confidence interval, 29.9-70.1%) and specificity of 100% (95% confidence interval, 75.3-100%). The kit was able to detect the NS1 antigen in CSF of individuals who had died of dengue. When used in combination with IgM, the detection rate rose to 92.3%. This study reports a method for rapidly detecting the dengue virus in CSF, thereby increasing the diagnosis of dengue fever cases with unusual neurological manifestations.
Subject(s)
Antigens, Viral/cerebrospinal fluid , Dengue Virus/immunology , Dengue/diagnosis , Enzyme-Linked Immunosorbent Assay , Reagent Kits, Diagnostic , Viral Nonstructural Proteins/cerebrospinal fluid , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, Viral/immunology , Child , Child, Preschool , Dengue/immunology , Female , Humans , Immunoglobulin M/blood , Immunoglobulin M/cerebrospinal fluid , Infant , Male , Middle Aged , Sensitivity and Specificity , Viral Nonstructural Proteins/immunology , Young AdultABSTRACT
An early diagnosis of Japanese encephalitis (JE) is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. The NS1 antigen has an advantage over IgM enzyme-linked immunosorbent assay (ELISA) for early confirmatory diagnosis of Japanese encephalitis virus (JEV) infection due to its proliferation on the surface of the host cells in the acute phase of infection. In this study, the development and evaluation of JE-specific NS1 antigen capture ELISA is described using high-affinity monoclonal antibody specific to the recombinant NS1 protein for early diagnosis of JE. The gene encoding NS1 protein was cloned and expressed in the pQE30UA expression vector followed by purification of the recombinant protein by affinity chromatography. A sandwich ELISA for antigen detection was developed using purified rabbit IgG antibody and mouse monoclonal antibody as the capture and detector antibody, respectively. The application of JE NS1 antigen ELISA for early diagnosis was evaluated with 120 acute phase sera and 80 CSF samples. The comparative evaluation of the JE NS1 antigen ELISA by real-time RT-PCR revealed 97% concordance with a sensitivity and specificity of 97% and 98%, respectively. The JE NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. These findings suggest that the JEV NS1 antigen capture ELISA may help early diagnosis of JE infection.
Subject(s)
Antibodies, Monoclonal , Encephalitis Virus, Japanese/immunology , Encephalitis, Japanese/diagnosis , Viral Nonstructural Proteins/immunology , Adolescent , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/blood , Antigens, Viral/immunology , Child , Child, Preschool , Encephalitis Virus, Japanese/genetics , Encephalitis Virus, Japanese/isolation & purification , Encephalitis, Japanese/immunology , Encephalitis, Japanese/virology , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Infant , Male , Mice , Mice, Inbred BALB C , RNA, Viral/genetics , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Viral Nonstructural Proteins/blood , Viral Nonstructural Proteins/cerebrospinal fluid , Viral Nonstructural Proteins/geneticsABSTRACT
Infection with hepatitis C virus (HCV) has become the most important public health problem in Egypt. HCV infection has been implicated in diseases of the central nervous system. Cerebrospinal fluid (CSF) and serum samples from 91 patients with meningitis (62 males and 29 females, mean age of 37 years) were investigated. Anti-HCV antibodies and HCV antigen were evaluated in patients CSF and serum using enzyme linked immunosorbent assay. The levels (mean +/- SD pg/ml) of Th1 cytokines (IFN-gamma and TNF-alpha) and Th2 interleukines (IL-10 and IL-4) were also determined. The anti-HCV antibodies were detected in high percentages both in CSF samples (71%) and in sera (90%). Also, the HCV antigen was detected in about 60% of tested CSF and serum samples. The levels of IFN-gamma and IL-10 cytokines were significantly higher (P < 0.05) in both serum and CSF of patients positive for HCV antigen than those negative. HCV antigen was detected in the CSF of meningitis patients with a significant upregulation of Th1 and Th2 responses. The high incidence of HCV infection may draw light on the etiological role of HCV in the pathogensis of meningitis diseases in our study group.
