Cerebrospinal fluid challenges for the diagnosis of herpes simplex infection in the central nervous system / Desafios do exame do líquido cefalorraquidiano para o diagnóstico de infecção por herpes simplex no sistema nervoso central

Abstract Herpes simplex virus (HSV) is a cause of a severe disease of the central nervous system (CNS) in humans. The demonstration of specific antibodies in the cerebrospinal fluid (CSF) may contribute to the retrospective neurological diagnosis. However, the commercial immunological tests for HSV infection are for use in serum samples. Objective: The aim of the present study was to adapt a commercial kit anti-HSV IgG used for serum samples to be performed with a CSF sample. Methods: Forty CSF specimens from 38 patients with suspected CNS HSV infection were serially diluted for detecting anti-HSV IgG by enzyme immunoassay (EIA). The same samples were also analyzed with the polymerase chain reaction (PCR). Results: The sensitivity of EIA test for HSV was 5% (dilution 1:40) and 65% (dilution 1:2) in CSF, and HSV DNA PCR was 15%. The combined analysis of EIA (dilution 1:2) and PCR increased the sensitivity up to 72.5%. The inflammatory CSF was associated with positive HSV PCR. Conclusions: We demonstrated the importance to adapt serological anti-HSV IgG EIA test for CSF assays to increase the accuracy of the analysis, considering the low concentration of specific antibodies in CSF.

Resumo O vírus herpes simples (HSV) é um dos agentes causadores de uma doença grave no sistema nervoso central (SNC) em humanos. A detecção de anticorpos específicos no líquido cefalorraquidiano (LCR) pode contribuir para o diagnóstico neurológico retrospectivo. Entretanto, os testes imunológicos comerciais são para uso em amostras de soro. Objetivo: Adaptar um kit comercial sorológico anti-HSV IgG para ser utilizado no de LCR. Metodos: Quarenta amostras de LCR de 38 pacientes com suspeita de infecção por HSV no SNC foram diluídas pesquisa de anticorpos anti-HSV IgG pelo método imunoenzimático (EIA). Além disso, as mesmas amostras também foram analisadas por reação em cadeia da polimerase (PCR). Resultados: A sensibilidade do teste EIA para o HSV consistiu em 5% (diluição 1:40) e 65% (diluição 1:2) no LCR, e o PCR do DNA do HSV, 15%. A análise combinada de EIA (diluição 1:2) e PCR aumentou a sensibilidade para 72,5%. Houve associação entre presença do LCR inflamatório e PCR positiva para HSV. Conclusões: Demonstramos a importância na adaptação previa do teste sorológico anti-HSV IgG EIA para ensaios do no LCR, a fim de aumentar a acuracia da análise, considerando a baixa concentração de anticorpos específicos no LCR.

Reaction of complement factors and proteasomes in experimental encephalitis.

Herpes simplex virus type 1 (HSV-1) encephalitis causes a deleterious inflammation and elevated intracranial pressure. As a step towards examining the origin of the inflammation, we here report the response of circulating proteasomes and complement factors in blood and cerebrospinal fluid (CSF) in rats infected with HSV-1. Infection was via the nasal route, with 1.1 × 104 plaque-forming units of HSV-1 strain 2762 given in one or both nostrils. A sandwich enzyme-linked immunosorbent assay was used to study the level of 26S proteasomes and their complex formation with complement factors 3 and 4. HSV-1 infection in the rat causes a complex formation between complement factors and proteasomes, which we designate compleasomes. In the first experiment, with HSV-1 given in both nostrils, compleasomes containing complement factors 3 and 4 increased significantly in both blood plasma and CSF. The concentration of proteasomes in plasma was similar in controls and infected rats (320 ± 163 vs. 333 ± 125 ng/ml). In the second experiment, with HSV-1 given in one nostril, CSF levels were 1 ± 1 ng/ml in controls and 56 ± 22 ng/ml in the HSV-1 group, whereas the total protein concentration in CSF remained the same in the two groups. The compleasome response was limited to CSF, with a highly significant difference between infected rats and controls (n = 11, p < 0.001). It was possible to mimic the reaction between proteasomes and complements 3 and 4 in vitro in the presence of ATP.

Development of recombinant antigen array for simultaneous detection of viral antibodies.

Protein microarrays have been developed to study antibody reactivity against a large number of antigens, demonstrating extensive perspective for clinical application. We developed a viral antigen array by spotting four recombinant antigens and synthetic peptide, including glycoprotein G of herpes simplex virus (HSV) type 1 and 2, phosphoprotein 150 of cytomegalovirus (CMV), Rubella virus (RV) core plus glycoprotein E1 and E2 as well as a E1 peptide with the optimal concentrations on activated glass slides to simultaneously detect IgG and IgM against HSV1, HSV2, CMV and RV in clinical specimens of sera and cerebrospinal fluids (CSFs). The positive reference sera were initially used to measure the sensitivity and specificity of the array with the optimal conditions. Then clinical specimens of 144 sera and 93 CSFs were tested for IgG and IgM antibodies directed against HSV1, HSV2, CMV and RV by the antigen array. Specificity of the antigen array for viral antibodies detection was satisfying compared to commercial ELISA kits but sensitivity of the array varied relying on quality and antigenic epitopes of the spotting antigens. In short, the recombinant antigen array has potential to simultaneous detect multiple viral antibodies using minute amount (3 µl) of samples, which holds the particularly advantage to detect viral antibodies in clinical CSFs being suspicious of neonatal meningitis and encephalitis.

Prolonged detection of herpes simplex virus type 2 (HSV-2) DNA in cerebrospinal fluid despite antiviral therapy in a patient with HSV-2-associated radiculitis.

Herpes simplex virus type 2 (HSV-2) can cause radiculo-myelitis as a neurological manifestation. We report a case of ongoing HSV-2 DNA positivity in the cerebrospinal fluid (CSF) of at least eight weeks under antiviral therapy with acyclovir in a highly immunocompromised hemato-oncologic patient with HSV-2-associated radiculitis. Upon admission, the patient presented with pain, leg paresis, and urinary incontinence, as well as pleocytosis in the CSF. Quantitative real-time PCR of the CSF at day 3 after admission revealed HSV-2 with a concentration of 2.0×10(5) copies/ml and treatment with acyclovir intravenously and prednisolone by mouth was started. Clinical symptoms resolved almost completely after approximately 3 weeks of antiviral therapy. However, CSF samples of day 12, 19, 26, 33, 39, 48 and 54 after admission showed a slow decline of HSV-2 DNA concentrations. HSV-2 DNA was still detectable (1.6×10(4) copies/ml) at day 54 after admission. Genotypic resistance testing showed, as far as available, no mutations indicative for acyclovir resistance. Since an increasing specific antibody index for HSV was observed, we speculate that the prolonged detectability of HSV-2 DNA in the CSF might not necessarily indicate ongoing viral replication but neutralized virus. Other hypotheses and the consequences on treatment are discussed. To our knowledge this is the first report about the long-term viral load kinetics of HSV-2 in the CSF of a patient with radiculitis under antiviral therapy, highlighting the need for further studies on HSV DNA kinetics in the CSF and their significance for an appropriate antiviral treatment.

Herpes simplex encephalitis following spinal ependymoma resection: case report and literature review.

Herpes simplex encephalitis (HSE) is a rare complication of neurosurgical procedures but must be considered in early deterioration of the postoperative patient. This is the first report of HSE following spinal cord tumor resection. A 65-year-old woman had C2-C5 laminectomy for subtotal resection of intramedullary ependymoma. Six days postoperatively she developed fever, vomiting and rapid decline in mental status. Brain MRI revealed enhancement of left insular cortex. Polymerase chain reaction on cerebrospinal fluid (CSF) identified herpes simplex virus type 1 (HSV-1) as the causal agent. Twenty-one days of acyclovir led to improvement. Three subsequent admissions to neurological intensive care unit were required for deterioration in mental status, including pneumonia, hydrocephalus and deep vein thromboses. Ventriculoperitoneal shunt (VPS), tracheotomy, percutaneous intravenous central catheter (PICC) line and percutaneous endoscopic gastrostomy (PEG) were placed. She was discharged to skilled nursing home care. Acyclovir is effective therapy against HSV, though outcomes may be poor even in optimally treated cases. Empiric treatment must be started even in the absence of serologic evidence of HSV infection if suspicion for HSE is high.

Negative association of Epstein-Barr virus or herpes simplex virus-1 with tumefactive central nervous system inflammatory demyelinating disease.

Central nervous system (CNS) demyelination has been suggested to be associated with infections caused by the Epstein-Barr virus (EBV) or herpes simplex virus (HSV)-1. CNS inflammatory demyelinating disease (IDD) rarely presents as a large lesion. We evaluated samples of serum and cerebrospinal fluid (CSF) by enzyme-linked immunosorbent assay to detect recent infection with these viruses and analyzed CSF and brain specimens by polymerase chain reaction (PCR) or immunohistochemical studies for evidence of these viruses in three patients with biopsy-proven CNS IDD. The results of PCR tests for EBV and HSV in CSF or brain specimens were negative. Elevated anti-EBV or -HSV antibody levels were not found in serum or CSF in any patient. Immunohistochemical studies showed that IDD lesions were negative for latent membrane protein (LMP)-1, Epstein-Barr nuclear antigen (EBNA)-2, and EBNA noncoding RNA (EBER)-1. These results suggest a negative association between CNS IDD and EBV or HSV.

Estudio experimental en ratas inoculadas intracerebralmente con líquido cefalorraquídeo de pacientes esquizofrénicos / Experimental study on intracerebral inoculated rats with cerebrospinal fluid taken from schizophrenic patients

Se hace un estudio mediante técnicas inmunoelectromicroscópicas en cerebros de cuatro ratas descendientes de padres inoculados intracerebralmente con el líquido cefalorraquídeo (LCR) de una paciente esquizofrénica hebefrénica, comparando sus resultados con un estudio en ratas controles. En un estudio anterior se habían encontrado alteraciones compatibles con la etiología por el herpes simplex hominis tipo I (HSV-1) en el cerebro de adultos esquizofrénicos, en fetos abortados utilizando la misma técnica de investigación, así como en embriones de pollo inoculados con LCR de pacientes esquizofrénicos. Los resultados obtenidos hasta el momento en este estudio pueden constituir un elemento más en favor de la transmisión de tipo vertical planteada para la enfermedad, así como su relación con el virus HSV-1. Estudios recientes en sueros obtenidos durante el embarazo de madres cuyos hijos padecieron esquizofrenia orientan hacia una etiología viral de la enfermedad y constituyen un elemento confirmatorio de la teoría del neurodesarrollo sobre la adquisición de la enfermedad antes del nacimiento por factores ambientales en el segundo trimestre de embarazo donde el herpes simplex se encuentra entre los candidatos más importantes.

A study is made by means of immuno-electron microscopic techniques in brains of four descending rats of parents inoculated intra-cerebrally with the cerebrospinal fluid (CSF) of a hebephrenic schizophrenic patient comparing its results with a study in control rats. In a previous study we have found alterations compatible with herpes simplex hominis type I (HSV1) virus etiology in the brain of schizophrenic adults, in aborted fetuses using the same research technique, as well as in chicken embryos inoculated with CSF from schizophrenic patients. The results obtained up to present in this study can constitute a further element favoring the vertical type transmission outlined for the illness as their relationship with HSV-1. Recent studies in serums obtained during pregnancy of mothers whose adult offspring suffered schizophrenia guide toward a viral etiology of the illness and they constitute a confirmatory element of the neurodevelopment theory about the acquisition of the illness before the birth due to environmental factors during the second pregnancy trimester where herpes simplex are among the most important candidates.

Incidence and pathogenesis of clinical relapse after herpes simplex encephalitis in adults.

OBJECTIVES: To study the occurrence of relapse of herpes simplex encephalitis (HSE) and to find out whether soluble activity markers in cerebrospinal fluid (CSF) indicate direct viral or immune- mediated events. METHODS: A consecutive series of 32 adult survivors of HSE were followed to determine the incidence of clinical relapse of HSE. Four patients had neurological deterioration interpreted as relapsing HSE. Four non-relapsing HSE cases were selected as matched controls. Fifty nine batched, paired CSF and serum samples from the eight HSE patients were analysed for soluble activity markers, predominantly cytokines and mediators (interferon-gamma, soluble CD8, tumour necrosis factor-alpha, and interleukin-10), amount of HSV-DNA and markers of glial and neuronal destruction (neurofilament protein, glial fibrillary acidic protein, S-100-beta, and neuron specific enolase). RESULTS: Relapse of HSE was diagnosed in 3 of 26 (12 %) acyclovir-treated patients (5 episodes during 6.1 years of followup) and in 1 of 6 vidarabine-recipients. All relapses occurred from 1 to 4 months after acute HSE, except for a second relapse after 3.3 years in one patient. Computer tomography at relapses revealed few abnormalities apart from those found during the primary disease. Intravenous acyclovir and corticosteroids were given for 7-21 days in all the relapse patients. All relapse patients seemed to recover to the pre-relapse condition. HSV-DNA was demonstrated in CSF in all patients during the acute stage but not in any of 13 CSF samples taken during relapse phases. The HSV viral load during the acute stage of HSE was not higher or of longer duration in the relapsing patients than in the non-relapsing HSE controls. The levels of sCD8 were increased in nearly all CSF samples tested with peaks of sCD8 at one month of acute HSE. In all episodes of relapse, sCD8 peaks were detected during the first week at high levels. CSF levels of neuron-specific enolase, S-100 and glial fibrillary acidic protein were markedly lower at relapse than at the acute stage of HSV-1 encephalitis. CONCLUSION: The lack of demonstrable HSV DNA in CSF, the lack of acute CSF signs and the lack of signs of neural and glia cells destruction indicate that a direct viral cytotoxicity is not the major pathogenic mechanism in relapse. Instead, the pronounced CSF proinflammatory immunological response and the relative lack of CSF anti-inflammatory cytokine IL-10 response suggest immunologically-mediated pathogenicity.

Herpes simplex virus type 2 as a cause of severe meningitis in immunocompromised adults.

We reviewed the clinical and demographic characteristics and outcomes for 13 immunocompromised patients with herpes simplex virus (HSV)-induced meningitis. Eleven patients were receiving chemotherapy for leukemia or lymphoma, and 10 had acquired immunodeficiency syndrome. Patients presented with acute febrile meningitis. The median white blood cell count at the onset of symptoms was 400 cells/mm3. Examination of cerebrospinal fluid (CSF) specimens showed lymphocytic meningitis, but activated lymphocytes and low glucose levels were both noted in 7 patients. HSV DNA was detected in all CSF specimens, and HSV type 2 was identified in 7. Eight patients had suspected HSV-associated mucocutaneous lesions at the time of meningitis onset. Six patients had initial radiculalgia, with sphincter involvement in 2. Eleven patients received intravenous antiviral therapy, but treatment was delayed for 6 patients. Two of the 6 patients for whom treatment was delayed developed encephalitis and died, whereas 2 others experienced persistent neurological symptoms. HSV-2 can cause severe meningitis in immunocompromised patients. Early recognition and treatment might improve the outcome of such infections.

[The role of intrauterine infection of the central nervous system with herpes simplex viruses in development of encephalopathies in infants]. / Znachenie vnutriutrobnogo infitsirovaniia tsentral'noi nervnoi systemy virusami prostogo gerpesa v razvitii éntsefalopatii u detei rannego vozrasta.

Intrauterine infections often affect the infant brain and cause meningoencephalitis and long-term encephalopathy. In this study we have analysed morphological changes in different parts of CNS of 22 deceased infants who had suffered from encephalopathy of different degree due to persistence of herpes simplex viruses. Morphological, immunofluorescent, serological methods were used. Antigens of herpes simplex viruses I and/or II were detected in the CNS of most examinees. Relevant antibodies were detected in the serum and cerebrospinal fluid. Intrauterine infection was confirmed by the presence of specific for herpes simplex viruses morphological changes in the placentas. Hydrocephaly, secondary microencephaly, false cysts, microgyria were found macroscopically. There were herpes simplex viruses specific histological changes such as nuclear hyperchromatosis, intranuclear basophilic and acidophilic inclusions and nonspecific changes (gliosis, productive vasculitis). The data demonstrate that intrauterine herpes simplex viruses persistence in the CNS is an important cause of infantile encephalopathy, specific and nonspecific changes indicate a chronic course of the infection and depend on the degree of clinical symptoms.

Herpes simplex virus infections of the central nervous system.

Herpes simplex virus (HSV) infections of the central nervous system (CNS) can occur within weeks after birth (neonatal HSV disease) or in childhood or adulthood [herpes simplex encephalitis (HSE)]. Most cases of neonatal HSV disease are caused by HSV type 2, whereas virtually all cases of HSE are caused by HSV type 1. Diagnostic advances made during the past decade include the application of polymerase chain reaction (PCR) technology to cerebrospinal fluid from patients with suspected HSV CNS disease to evaluate for the presence of HSV DNA. Although not foolproof, PCR is a powerful diagnostic tool that has supplanted brain biopsy as the modality of choice for diagnosing HSV CNS disease, in no small part because of the invasiveness of brain biopsy. PCR also can provide information regarding the therapeutic response to antiviral therapy. Efforts made during the past decade to improve the outcome of HSV CNS disease have focused on increased doses of intravenous acyclovir administered for longer durations of time. Although advances have been achieved, morbidity and mortality rates from neonatal HSV disease and HSE remain unacceptably high.

Monocyte chemoattractant protein-1 enhances HSV-induced encephalomyelitis by stimulating Th2 responses.

Monocyte chemoattractant protein (MCP)-1 has a pathogenic role in herpesvirus-induced encephalomyelitis (HSM). Anti-MCP-1 antibody greatly decreased HSM severity in mice infected with herpes simplex virus type 2 (HSM mice), compared with its effect in control HSM mice treated with rabbit immunoglobulin. HSM severity was markedly enhanced in mice previously treated with a mixture of interleukin (IL) 4 and -10. In response to stimulation with antigen, HSM mouse cells isolated from cerebrospinal fluids (CSF cells) produced IL-4 in culture fluids; however, IL-4 production decreased in CSF cells derived from HSM mice previously treated with anti-MCP-1 antibody. A macrophage population isolated in CSF cells from HSM mice (CSF-Mphi) produced MCP-1 in culture fluids. In response to stimulation with herpesvirus antigen, a population of T cells isolated from CSF cells from HSM mice (CSF-T cells) produced IL-4 into their culture fluids, although MCP-1 was not produced by CSF-T cells stimulated by this antigen. IL-4 production by CSF-T cells was markedly enhanced when they were stimulated with viral antigen in the presence of murine recombinant MCP-1 (rMCP-1). Furthermore, IL-4 was produced in naive splenic T cells cocultured with CSF-Mphi. These results indicate that the severity of HSM is influenced by MCP-1, which stimulates Th2 responses.

A single tube PCR assay for simultaneous amplification of HSV-1/-2, VZV, CMV, HHV-6A/-6B, and EBV DNAs in cerebrospinal fluid from patients with virus-related neurological diseases.

Cerebrospinal fluid (CSF) specimens from 27 patients with encephalitis, meningitis, and other neurological diseases were studied for the presence of herpes simplex virus types 1 and 2 (HSV-1/-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), human herpesviruses 6A and 6B (HHV-6A/-6B) and Epstein-Barr virus (EBV) DNA using the polymerase chain reaction (PCR) method. The DNAs were amplified using two sets of consensus primer pairs in a single tube, bringing simultaneous amplification of the herpesviruses. The PCR products were analyzed by agarose gel electrophoresis, and Southern blot hybridization with virus-type specific probes, thus allowing discrimination between the different types of herpesviruses to be made. Each virus-specific probe was highly specific for identifying the PCR product. Thirty CSF specimens from 13 patients with encephalitis and 10 specimens from 10 patients with meningitis, respectively, were examined using this method. Eight patients with encephalitis and six with meningitis were positive for different herpesviruses, including patients with coinfections (HSV-1/-2 and VZV, VZV and CMV). Among four CSF specimens from four patients with other neurological disorders, dual amplification of CMV and EBV was present. Since identification of the types of herpesviruses in this system requires a very small amount of CSF, and is completed with one PCR, it is useful for routine diagnosis of herpesvirus infections in diagnostic laboratories. The viruses responsible for central nervous system infection are easily detected with various coinfection and serial patterns of herpesviruses, by this consensus primer-based PCR method. This may give an insight into the relationship between virus-related neurological diseases (VRNDS) and herpesvirus infections.

Positive CSF HSV PCR in patients with GBM: a note of caution.

Because two patients with temporal lobe glioblastomas had herpes simplex (HSV) DNA detected in CSF using PCR at the time of their presentation, we reviewed our laboratory's experience and performed PCR on a bank of 159 frozen CSF samples from patients with glioblastoma multiforme and other neurologic disorders. Based on the inability to detect HSV in any other tumor sample, we conclude that the positive HSV PCR in our two index patients most likely represented false-positive results. A diagnosis of HSE should not be made by PCR alone when the clinical presentation is atypical.

[Virus encephalitis with symptomatic Parkinson syndrome, diabetes insipidus and panhypopituitarism]. / Virusenzephalitis mit symptomatischem Parkinsonsyndrome Diabetes insipidus und Panhypopituitarismus.

Virusencephalitis is characterised by clinical symptoms of a parenchymatous inflammation. In addition, early mental status changes often occur as a result of virusencephalitis, beside focal neurological deficiencies, epileptic seizures, cerebral compression, even coma. Other pathological manifestations of virusencephalitis are disturbances of the neurohumoral and the endocrine system, which are often recognised and treated too late. This case report describes symptoms, treatment, and complications of a 76 year old female in-patient, who was diagnosed with virusencephalitis. The number of lymphocytes in the cerebrospinal fluid was increased to 30 cells per microliter, liquor albumin was 1705 mg/l, liquor sugar was 53 mg/dl and liquor lactat was 1.9 mmol/l. IgM antibodies against herpes viruses were found in the cerebrospinal fluid and distinct contrasting foci were found near the mammillary bodies, hypothalamus, tractus opticus, hypophyseal stalk and right parahippocampal in the magnetic resonance imaging of the head, indicating a focal herpes simplex encephalitis. Within seven days, the following symptoms developed: akinetic parkinsonian syndrome, central diabetes insipidus with hypernatremia and polyuria (6 l/die), hypothyreosis, adrenal insufficiency with adynamia, sopor, hypotension and even hypophyseal coma. Panhypopituitarism was diagnosed after measuring the basal hormone levels (ACTH, TSH, FT3, FT4, Cortisol, Prolactin, LH, FSH, ADH) and conducting the pituitary stimulation test. The severeness of all symptoms was slightly improved after substitution with antidiuretic hormone at 0.4 microgram/die and administration of hydrocortisone at 50 mg/die. Administration of amantadine sulphate at 0.6 g/die and L-dopa at 187.5 mg/die for 14 days resulted in a complete regression of the parkinsonism. After administration of aciclovir at 2.25 g/die for 21 days a complete regression of the clinical symptoms could be reached in connection with a decrease of 90% in number and size of cerebral contrasting foci in the magnetic resonance imaging of the head. Three month after therapy, clinical examination and blood serum analysis revealed persistent panhypopituitarism. The present case report is the first description of a viral infection on of the central nervous system (CNS) in combination with parkinsonism, diabetes insipidus, persistent panhypopituitarism and hyperprolactinemia. Early treatment of viral infections of the brain can improve a patient's prognosis dramatically. Early determination and early treatment of a patient's neurohumoral parameters is therefore critical to prevent or reverse early mental status changes like attention disturbances, alterations of personality and behavior, apathy, and slowed cognition.

A duplex PCR assay for detection and genotyping of Herpes simplex virus in cerebrospinal fluid.

A duplex polymerase chain reaction (PCR) assay for the detection and genotyping of Herpes simplex virus (HSV) 1 and 2 from cerebrospinal fluid (CFS) of infants was developed. The glycoprotein D (gD) gene of HSV was selected as a target for amplification. The assay is highly specific, sensitive and reproducible. Herpes simplex virus detection is performed by agarose gel electrophoresis and Southern blot using a chemiluminescent probe. The probe hybridizes to sequences common to both HSV-1 and 2. A DNA fragment of HSV gD gene was cloned and used as positive control and to determine the specificity and sensitivity of the assay. The PCR assay is user-friendly and unambiguously differentiates in one-step both herpes virus strains. The assay is useful to screen CFS specimens from infants exposed to HSV during birth and at risk of developing encephalitis.

Development of a high-throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples.

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 10(8) copies of HSV DNA/20 microl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

[Immunoenzyme analysis and polymerase chain reaction in the diagnosis of an intrauterine infection in newborn infants with cerebral lesions]. / Immunofermentnyi analiz i polimeraznaia tsepnaia reaktsiia v diagnostike vnutriutrobnoi infektsii u novorozhdennykh s tserebral'nymi porazheniiami.

The comparative evaluation of the diagnostic value of the polymerase chain reaction (PCR) and the enzyme immunoassay (EIA) in the detection of intrauterine infection (IUI) in 48 newborn infants with cerebral lesions was made. Tests for the presence of the infective agents of IUI, most frequently occurring in the region (Cytomegalovirus, Herpes simplex virus, Chlamydia trachomatis), were carried out. The levels of serum IgA, IgG and IgM were evaluated in the course of the primary screening of IUI. Laboratory samples for PCR from infants with IUI were selected at random. The study demonstrated that in PCR the frequency of positive results was significantly greater than in EIA.

[Herpes simplex and lymphocytic choriomeningitis viruses in infections of the central nervous system--clinical and cerebrospinal fluid characteristics]. / Virusi herpesa simpleks i limfocitarnog horiomeningitisa u infekcijama centralnog nervnog sistema--klinicke i likvorske karakteristike.

INTRODUCTION: A great number of various viruses are stated as the cause of acute infections and damages of the central nervous system. In most cases these are minor damages which exhibit as meningeal syndrome and a specific finding in the cerebrospinal fluid. According to the dominant location, central nervous system infections can take a form of meningitis, encephalitis or myelitis. Since the inflammatory process of the meninges can not be separated from the inflammatory process of the brain, we usually speak of meningoencephalitis. The etiological diagnosis of meningitis and encephalitis is established by isolating the virus from the cerebrospinal fluid and by finding the presence of the specific antibodies in the blood and in the cerebrospinal fluid. The most common causes of the viral meningitis are Enteroviruses, the Mumps virus, Arthropode borne viruses, the Herpes viruses, Adeno viruses and the Lymphocytic choriomeningitis virus. The aim of our study was to establish the correlation between the clinical features and immunological and cerebrospinal fluid changes and the degree of the damage to the blood-brain barrier during the infections of the central nervous system, caused by the Herpes Simplex virus and the Lymphocytic choriomeningitis virus. MATERIAL AND METHODS: From a group of 103 patients, who had been treated for viral meningitis and meningoencephalitis, a group of 27 patients with established specific viral etiology--Herpes Simplex virus and Lymphocytic choriomeningitis virus, had been taken into the account. Herpes Simplex infection had been proven by the complement binding reaction and the neutralisation test of the even samples of serum. The diagnosis of Lymphocytic choriomeningitis was confirmed by the immunofluorescence test of the pharynx swabs and cerebrospinal fluid. The clinical features, such as body temperature, encephalitic signs, and electroencephalographic findings had been followed and compared. RESULTS: Herpes Simplex infection had been found in 20 patients, Lymphocytic choriomeningitis had been proven in 7 patients. All the patients had increased body temperature. Only four of the patients exhibited encephalitic signs, all infected by the Herpes Simplex virus. Patients from the Herpes Simplex group showed various degrees of consciousness disturbances, ranging from somnolence to coma, while the Lymphocytic choriomeningitis patients exhibited none. Higher pleocytosis and protein level had been found in the Lymphocytic choriomeningitis group. DISCUSSION: Viral diseases of the central nervous system are the result of the direct damage of the brain and meninges by the virus and immunological processes. Herpes Simplex meningitis usually has a good prognosis. Lymphocytic choriomeningitis has longer course of the disease and exhibits more severe clinical features. CONCLUSION: In cases of the central nervous system infections, caused by Herpes Simplex virus or Lymphocytic choriomeningitis virus, the correlation between the severeness of clinical features and the degree of damage of the blood-brain barrier, the level of pleocytosis and the increase of the cerebrospinal fluid proteins had been established.