First DFT-supported point of care and novel electrochemical biosensing: Determination of yellow fever NS1 antibody in human plasma.

In our study, we developed a point of care electrochemical biosensing platform based on the functionalized cysteine-positioned gold electrode to diagnose yellow fever disease from human plasma samples. The developed platform underwent characterization through diverse methods encompassing cyclic voltammetry, electrochemical impedance spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, and density-functional theory. The capacitive interaction between yellow fever virus non-structural antigen and antibody gave a cathodic signal at approximately -260 mV, and increased in proportion to the amount of non-structural antibody. The created electrochemical biosensor has an ability to detect 96 ag/mL of the yellow fever non-structural antibody with an extensive analytical range varied from 0.1 fg/mL to 1 µg/mL. The interference effects of various substances that could be found in human plasma, and the performance of the method were examined from the point of recovery and relative standard deviation for human plasma samples; hereby, the results confirmed the unprecedented selectivity and accuracy of the proposed method.

Oriented Immobilization of Single-Domain Antibodies Using SpyTag/SpyCatcher Yields Improved Limits of Detection.

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

Development and Characterization of Double-Antibody Sandwich ELISA for Detection of Zika Virus Infection.

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillain-Barré syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early viral infection. Furthermore, these methods require the extraction of RNA, which is easy to contaminate. Nonstructural protein 1 (NS1) is an important biomarker for early diagnosis of the virus, and the detection methods associated with the NS1 protein have recently been reported. The aim of this study was to develop a rapid and sensitive detection method for the detection of the ZIKV based on the NS1 protein. The sensitivity of this method is 120 ng mL-1 and it detected the ZIKV in the supernatant and lysates of Vero and BHK cells, as well as the sera of tree shrews infected with the ZIKV. Without the isolation of the virus and the extraction of the RNA, our method can be used as a primary screening test as opposed to other diagnosis methods that detect the ZIKV.

Cerebral vasculitis and lateral rectus palsy - two rare central nervous system complications of dengue fever: two case reports and review of the literature.

BACKGROUND: Dengue fever is a common mosquito-borne viral illness with a clinical spectrum ranging from a simple febrile illness to potentially life-threatening complications such as dengue hemorrhagic fever and dengue shock syndrome. Dengue infection can affect many organs, including the central nervous system. The neurological manifestations reported in dengue infections are meningitis, encephalitis, stroke, acute disseminated encephalomyelitis, and Guillain-Barré syndrome. CASE PRESENTATION: We report the cases of two interesting patients with confirmed dengue infection who presented with complications of possible central nervous system vasculitis and cranial nerve palsy. The first patient was a 53-year-old previously healthy Singhalese woman who developed acute-onset slurring of speech and ataxia with altered sensorium 1 day after recovery from a critical period of dengue hemorrhagic fever. Subsequent investigations revealed evidence of encephalopathy with brainstem ischemic infarctions. Her clinical picture was compatible with central nervous system vasculitis. She was treated successfully with intravenous steroids and had a full functional recovery. The second patient was a middle-aged Singhalese woman who had otherwise uncomplicated dengue infection. She developed binocular diplopia on day 4 of fever. An ocular examination revealed a convergent squint in the left eye with lateral rectus palsy but no other neurological manifestation. CONCLUSIONS: Central nervous system vasculitis due to dengue infection is a very rare phenomenon, and to the best of our knowledge, only one case of central nervous system vasculitis has been reported to date, in a patient of pediatric age. Cranial nerve palsy related to dengue infection is also rare, and only a few cases of isolated abducens nerve palsy have been reported to date. The two cases described in this report illustrate the rare but important central nervous system manifestations of dengue fever and support the fact that the central nervous system is one of the important systems that can be affected in patients with dengue infection.

HCV nonstructural protein 4 is associated with aggressiveness features of breast cancer.

BACKGROUND: Hepatitis C virus (HCV) has the lymphotropic feature that is supposed to be the reason of related extrahepatic manifestation. HCV viral oncoproteins may participate in the regulation of some gene expression that has been implicated in tumorigenesis. Our aim is to evaluate the HCV-NS4 circulating levels in breast cancer (BC) and to investigate its relation with BC tumor aggressiveness. METHODS: This study was performed among 158 Egyptian women (120 with BC and 38 with benign breast diseases). ELISA was used for detection of anti-HCV antibodies, HCV-NS4, fibronectin, and CA 15-3. RESULTS: No association between HCV detection in this group of BC patients (27.5% in BC vs. 23.7% in breast benign diseases, P = 0.687). Among HCV-infected patients, the mean HCV-NS4 serum level in BC was significantly higher than benign group (61.7 µg/mL vs. 33.9 µg/mL, P = 0.0005). Fibronectin levels were higher (P = 0.014) in patients infected with HCV than noninfected BC patients. Elevated HCV-NS4 levels were associated with tumor severity features like large size, late stages, high grades, and infiltrated lymph nodes. The elevated levels of HCV-NS4 (> 40 µg/mL) yielded an estimated odds ratio (95% confidence intervals) of 2.5 (0.98-6.36), 1.2 (0.44-3.33), 1.9 (0.53-7.00), and 2.5 (0.87-7.33) for developing large size, late stages, high grades, and infiltrated lymph nodes, respectively. Interestingly, HCV-NS4 levels significantly correlated with other BC tumor marker like CA15-3 (r = 0.535; P = 0.0009) and fibronectin (r = 0.432; P < 0.0001). CONCLUSIONS: HCV-NS4 appears to be associated with BC progression features. Oncologists treating such BC patients should consider HCV screening to enable the early identification and to prevent progression of the disease.

Perinatal transmission of hepatitis C antigens: envelope 1, envelope 2 and non-structural 4.

BACKGROUND: Perinatal exposure to hepatitis C virus (HCV) antigens during pregnancy may affect the developing immune system in the fetus. We aimed to study the perinatal transmission of HCV structural and non-structural antigens. METHODS: Sera from 402 pregnant mothers were tested for anti-HCV antibody and HCV RNA. HCV antigens were determined in sera from 101 HCV-infected mothers and their cord blood. RESULTS: In both serum and cord blood samples, HCV NS4 (non-structural 4) at 27 kDa, E1 (envelope 1) at 38 kDa and E2 (envelope 2) at 40 kDa were identified, purified and quantified using western blotting, electroelution and ELISA. Maternal sera and neonate cord blood samples had similar detection rates for NS4 (94.1%), E1 (90.1%) and E2 (90.1%). The mean maternal serum levels (optical density, OD) of HCV NS4 (0.87 ± 0.01), E1 (0.86 ± 0.01) and E2 (0.85 ± 0.01) did not differ significantly (p > 0.05) from those of neonatal cord blood (0.83 ± 0.01, 0.87 ± 0.01 and 0.85 ± 0.01, respectively). Also, strong correlations (p < 0.0001) were shown between sera and cord blood sample levels of HCV NS4, r = 0.77; E1, r = 0.76 and E2, r = 0.80. The vertical transmission of these antigens in vaginal delivery did not differ significantly (p > 0.05) from those in caesarean section. CONCLUSIONS: These findings indicate that vertical transmission of HCV NS4, E1 and E2 antigens was very high. Thus, exposure to these antigens may influence the developing immune responses to natural infection or future vaccination.

Seronegative hepatitis C virus infection in patients with lymphoproliferative disorders.

It has been reported that hepatitis C virus (HCV) RNA may be present in serum and/or lymphoid cells in the absence of specific circulating antibodies. The current study analysed seronegative HCV infection in patients with lymphoproliferative disorders. We studied 77 anti-HCV-negative patients (45 male and 32 female, mean age 54.8 ± 14.2 years) with various lymphoproliferative disorders. HCV-RNA was detected by RT-PCR in plasma, peripheral blood mononuclear cells (PBMC) and bone marrow. Furthermore, the presence of viral nonstructural protein 3 (NS3) was determined in PBMC and bone marrow by immunostaining. HCV-RNA was detectable in at least one compartment in 27 (35.1%) patients. Viral RNA was found in bone marrow in 22 patients (28.6%), in PBMC in 13 (16.9%) and in plasma in 10 (13%) patients. In nine patients, evidence of infection was confined to the bone marrow compartment. Viral load in HCV-RNA-positive plasma ranged from 15 to 1.17 × 10(3) IU/mL. NS3 was detected in all but two HCV-RNA-positive bone marrow samples and in all but one HCV-RNA-positive PBMC samples. All 27 HCV-RNA-positive patients remained anti-HCV-negative when tested again after 6-12 months, but only four remained HCV-RNA positive. In conclusion, among patients with lymphoproliferative disorders, HCV can be present in plasma, PBMC and bone marrow despite the lack of circulating specific antibodies. Further studies are required to analyse the phenomenon of seronegative infection and to determine whether such patients are infectious.

The current hepatitis C virus prevalence in China may have resulted mainly from an officially encouraged plasma campaign in the 1990s: a coalescence inference with genetic sequences.

In this study, we investigated hepatitis C virus (HCV) molecular epidemiology and evolutionary dynamics. Both E1 and NS5B sequences were characterized in 379 of 433 patients in southern China and classified into five major subtypes: 1b in 256 patients, 6a in 67 patients, 2a in 29 patients, 3a in 14 patients, and 3b in 13 patients. Using the E1 sequences obtained, along with those from other studies using samples from China, we inferred the HCV epidemic history by means of coalescence strategies. Five Bayesian skyline plots (BSPs) were estimated for the five subtypes. They concurrently highlighted the rapid growth in the HCV-infected population size from 1993 to 2000, followed by an abrupt slowing. Although flanked on both sides by variable population sizes, the plots showed distinct patterns of rapid HCV growth. Coincidently, 1993 to 2000 was a period when contaminated blood transfusions were common in China due to a procedural error in an officially encouraged plasma campaign. The abrupt slowing in 1998 to 2000 corresponded to the central government outlawing paid blood donations in 1998. Using a parametric model, the HCV population growth rates were estimated during 1993 to 2000. It was revealed that the 6a rate was the highest, followed by those of 1b, 2a, 3b, and 3a. Because these rates differed significantly (P < 1e-9) from each other, they may help explain why 6a is increasingly prevalent in southern China and 1b is predominant nationwide. These rates are approximately 10-fold higher than those reported elsewhere. These findings suggested that during the plasma campaign, certain barriers to efficient viral transmission were removed, allowing wide HCV dissemination.

[Case report of the available of interhenotypic recombinant in chronic hepatitis C: the algorithm of individualization of the antiviral therapy].

UNLABELLED: In this particular case report (in vivo the persistence of HCV intergenotipic recombinant form /2/1b/ peripheral blood mononuclear cells of patient) is an example of optimizing the standard antiviral therapy (PegIntron + Rebetol). In this paper we report a patient infected by HCV 1b and, probably, recombinant 2/1b that is detected in peripheral blood mononuclear cells (PBMC). CASE DESCRIPTION: Patient S., male, 31 years old admitted in January 2009. HCV viral load in serum before treatment 9,630,000 IU/ml. HCV genotyping by sequencing 5'UTR and NS5A. According to phylogenetic analysis NS5A belongs to 1b (sera and PMBC), 5'UTR from serum to 1b, from PBMC to genotype 2. Due to discordant results recombinant 2/1b in PBMC can be suspected. NS5A interferon sensitivity determining region (ISDR) contains mutation R2218H. Laboratory: ALT 71 U/l, AST 62 U/l, GGT 36 U/l. Liver biopsy: HAI 18, fibrosis 3. Immunohistochemically HCV NS3 was detected in lobules and tracts. Elevated CD16 and CD20 was found in lymphoid follicules of portal tracts. Patient received treatment with pegintron (1.5 mg/kg BW) plus ribavirin (1000 mg/day) for 48 weeks. Virological and biochemical response were achieved on 12 wk and remained until the end of treatment and during follow-up. Liver biopsy after treatment: HAI 6, fibrosis II. Immunohistochemically NS3 was still detected in lobules and tracts, CD16 and CD20 decreased in portal tracts. We also discuss the justification of the selected option treatment strategy (reduced time-course of combination antiviral therapy /28 weeks/ and supplementation of course of antiviral therapy without interferon: a combination of interferon inducer /Cycloferon/ or antiviral drugs /Rebetol/ for another 12 weeks.

Evaluación del sistema diagnóstico SD Dengue Duo para la detección de la proteína NS1 y los anticuerpos IgM e IgG anti-dengue / Evaluation of the SD Dengue Duo diagnosis system for detection of NS1 protein and IgM and IgG dengue antibodies

Introducción: el sistema comercial SD Dengue Duo (Standard Diagnostics) es una prueba rápida inmunocromatográfica que detecta la proteína NS1 del virus dengue y los anticuerpos anti-dengue IgM e IgG de forma simultánea. Objetivo: evaluar las características funcionales y operacionales del sistema comercial para la detección de los diferentes marcadores virológicos y serológicos. Métodos: se utilizó un panel constituido por 161 muestras de suero, 113 procedentes de pacientes con un diagnóstico clínico y serológico confirmado a cualquiera de los 4 serotipos del virus dengue y 48 negativas. Todas las muestras fueron procesadas por el sistema SD Dengue Duo y por las técnicas Platelia Dengue NS1 Ag, ELISA de captura IgM y el ELISA de inhibición utilizadas como referencia. Resultados: el sistema evaluado mostró una sensibilidad de 57,75 por ciento para la proteína NS1, se observaron falsos negativos en muestras colectadas a partir del quinto día de iniciados los síntomas en casos con infección secundaria. La detección de anticuerpos IgM mostró una sensibilidad de 96,0 por ciento y especificidad de 98,4 por ciento. Se encontró una alta concordancia (95,7 por ciento) al clasificar el tipo de infección. En el estudio global de los 3 marcadores la sensibilidad del sistema se incrementó hasta 100 por ciento. Conclusiones: el SD Dengue Duo es un método simple, rápido, fácil de ejecutar, el cual no requiere de equipamiento adicional; tiene la ventaja de poder ser utilizado para muestras, tanto de fase aguda como en la fase convaleciente y pudiera ser una alternativa para el diagnóstico del dengue en aquellos laboratorios que no cuenten con facilidades para ello.

Introduction: SD Dengue Duo (Standard Diagnosis) commercial kit is an immunochromatographic rapid test that detects NS1 protein and IgG/IgM dengue antibodies simultaneously. Objective: to evaluate the operational and functional characteristics of this system for the detection of virological and serological markers. Methods: sera panel was made up by 161 samples, 113 from patients with clinically and serologically confirmed dengue caused by any of the four dengue virus serotypes and 48 negative samples. All these samples were tested by SD Dengue Duo Kit and by Platelia Dengue NS1 Ag, IgM Capture ELISA and ELISA Inhibition Method used as reference assays. Results: the evaluated kit showed a 57.75 percent sensitivity for the detection of NS1 protein, false negatives were detected in samples collected 5 days or more after fever onset in secondary infection cases. IgM detection showed 96.0 percent sensitivity and 98.4 percent specificity. Furthermore, high agreement (95.7 percent) in classifying dengue infection types (primary or secondary infections) was observed. The global study of the 3 markers, the sensitivity rose to 100 percent. Conclusions: SD Dengue Duo is a simple, easy and rapid assay; it does not require additional equipment, can be used for acute and convalescence serum samples and offers a good alternative for dengue diagnosis in those laboratories where a complete dengue virus diagnosis is difficult to perform.

Severe nonfebrile dengue infection in an adolescent after postoperative kidney transplantation: a case report.

We herein have reported a case of severe nonfebrile dengue infection complicated with refractory pancytopenia and a large perinephric hematoma with shock in a 16-year-old adolescent during the early postoperative period after kidney transplantation. After the diagnosis of end-stage renal disease she underwent living-related kidney transplantation. Thirteen days after successful transplantation, she exhibited a notable amount of ascites, bilateral pleural effusions, thrombocytopenia, and increased hemoglobin without pre-existent fever. Further investigation revealed positive dengue nonstructural protein 1 antigen (dengue NS1 Ag) and dengue virus serotype 1 by a reverse transcriptase-polymerase chain reaction (RT-PCR) in the patient's serum. She exhibited hemophagocytic syndrome, manifested by refractory pancytopenia and refractory anemia resulting in hypovolemic shock and acute graft failure on day 28 posttransplantation. The anemia was attributed to a large hematoma around the transplanted kidney requiring surgical evacuation of clotted blood. Postoperatively, she gradually recovered with resolution of thrombocytopenia and excellent graft function. Persistent dengue antigenemia and viremia was shown by dengue NS1 Ag and RT-PCR of dengue serotype-1. The viremia was present longer than the dengue antigenemia. Dengue-specific immunoglobulin M (IgM) and IgG by enzyme-linked immunosorbent assay confirmed the primary dengue infection and evidence of a recent donor dengue infection.

Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization.

Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non-structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV-infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs. Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (P = 0.0511 and 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (P = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for one woman, while statistical methods were consistent with PBMC compartmentalization for six women. Evidence of compartmentalization within a non-structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence.

HCV non-structural protein 3 and HCV RNA genome in non-Hodgkin lymphoma and transition of the serum HCV RNA level: a retrospective analysis in one institution.

There have been various reports on the association of hepatitis C virus (HCV) infection with B lymphocyte proliferative disorders, such as non-Hodgkin lymphoma (NHL). We experienced a case (Case 1) of anti-HCV antibody (HCV-Ab)-positive NHL in which HCV nonstructural protein 3 (NS3) expression was observed in lymphoma tissue at the time of recurrence and in which the serum HCV RNA level exhibited a transient increase prior to recurrence. We investigated the HCV RNA genome in lymphoma tissue in Case 1, and it could be detected at recurrence. We also investigated HCV NS3 protein expression in lymphoma tissue and changes in serum HCV RNA level during the clinical course in four other cases of HCV-Ab-positive NHL treated in our hospital. We examined lymphoma tissues for HCV NS3 protein expression in four of the five cases, but it was not identified except for in Case 1 at recurrence. In three cases with no recurrence, serum HCV RNA levels showed a tendency to decrease after completion of chemotherapy and became stable thereafter. Further studies are necessary to clarify the association between serum HCV RNA and the onset and exacerbation of NHL.

Immunochemical identification and partial characterization of a native hepatitis C viral non-structural 4 antigen in sera of HCV infected patients.

BACKGROUND: The identification of native HCV antigens may prove very useful in the diagnosis and early treatment of HCV infection. Here, we aimed to identify and partially characterize a native HCV-NS4 antigen. METHODS: The western blot, ELISA and immunohistochemical staining were used to identify the native antigen in sera and liver biopsies of HCV serotype 4 infected patients. RESULTS: The native NS4 antigen was identified in serum at 27-kDa molecular weight, in addition to a lower approximately 21-kDa antigen. The purified HCV antigen showed a polypeptide band at 27-kDa when analyzed by silver stained SDS-PAGE and a single peak at 7.6 min by capillary zone electrophoresis. The immunostaining pattern of hepatocytes was cytoplasmic with mainly coarse granular and diffuse pattern based on specific rabbit antisera to the native HCV antigen. A highly significant correlation (r=0.797, p<0.0001) was shown between serum concentrations of the HCV-NS4 antigen and HCV-RNA. Also, antigen detection rates were increased (p<0.05) with the progression of liver disease. CONCLUSION: A native HCV-NS4 antigen was identified and partially characterized as 27-kDa protein and the NS4 antigenemia based ELISA test can serve as a useful addition to HCV diagnostic methods, especially under field condition.

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Piezoelectric immunochip for the detection of dengue fever in viremia phase.

The global prevalence of dengue fever has grown so dramatically in recent years that it is endemic in more than 100 countries and has become a major international public health concern. Moreover, since the flu-like symptoms that accompany dengue fever are atypical and varied, the detection procedures currently used to identify it are cumbersome and time-consuming, making early stage epidemiological control and effective medical treatment of this epidemic almost impossible. In this study, a QCM-based detection system was developed in which two monoclonal antibodies against dengue E and NS-1 protein, respectively, were control orientated immobilized on QCM via protein A to produce an immunochip. Various sample pretreatment procedures were evaluated to ascertain the most suitable combination, and both the simulating samples and the clinical specimen were examined by the immunochip. The results revealed that the cibacron blue 3GA gel-heat denature (CB-HD) method was the most effective sample pretreatment technique. Due to the complex composition of the serum, the immunochip could only effectively quantify dengue viral antigens in a 1/1000 untreated simulated sample. With the help of the CB-HD method, the dilution folds were found to capable of being reduced from 1000 to 100, and the detection limit lowered to 1.727 microg/ml (E protein) and 0.740 microg/ml (NS-1 protein) in the original sample. While the cocktail immunochip could not quantify both antigens separately, the higher signal level rendered it a more effective qualification tool for suspect screening. Moreover, the results of the analysis of clinical specimens also proved the ability and future potential of cocktail immunochip in discriminating dengue-positive cases from negative serum specimens in the viremia phase.

Detection of antibody to the foot-and-mouth disease virus (FMDV) non-structural polyprotein 3ABC in sheep by ELISA.

The specificity and sensitivity of an ELISA for detecting IgG to the 3ABC non-structural protein of foot-and-mouth disease (FMD) virus was evaluated in FMD naive, aerosol-infected, aerosol plus direct contact infected and field-exposed sheep. All 12 sheep that were experimentally infected without prior vaccination seroconverted in the test, although fewer field sera from FMD-exposed sheep were scored seropositive compared to test results for structural protein antibodies. The 3ABC test specificity was 98 or 100% according to whether sera reacting in the doubtful range were scored as positive or negative. The test was then used to investigate the antibody response of sheep vaccinated against FMD and exposed to the virus by an aerosol challenge 4-14 days later. The response of individual animals varied. Whether immunised with high or low doses of vaccine, the development of 3ABC antibody was most likely in sheep from which live virus was recovered at or beyond 9 days post-challenge. Non-structural responses were also more frequent in animals from which multiple incidences of live FMD virus isolation (perhaps more indicative of true virus replication) were demonstrated.

Immunodetection of a hepatitis C virus (HCV) antigen and Thl/Th2 cytokines in cerebrospinal fluid of meningitis patients.

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.