Rapid diagnosis of tick - borne encephalitis by immunofluorescence assay of specific serum IgM antibodies.

The indirect IF technique, using suspensions of TBE virus infected and uninfected PS cells as antigen-containing substrate, furnishes a rapid and practical test making possible the detection of specific IgM class serum antibodies in the initial stage of clinically manifest TBE. It enables early confirmation of diagnosis already in the acute phase of the disease and thus it can be instrumental in differential diagnosis and rational therapy, e.g., the administration of specific hyperimmune gamma-globulin.     

The characteristics of detecting the tick-borne encephalitis virus antigen in the ELISA and indirect hemagglutination reaction by means of scanning electron microscopy

The surface of polystyrene plates was studied at different stages of the enzyme immunoassay (EIA) and the passive hemagglutination (PHA) test by the method of scanning electron microscopy in the detection of tick-borne encephalitis (TBE) virus antigen. The study revealed that in the process of EIA larger antigens were washed away from the plate surface. The objects detected on the polystyrene surface were identified as conglomerations of the virions of TBE virus, but whole virions were shown to play no decisive role in EIA. The conclusion was made that, due to some specific features of this method, EIA was more sensitive in reaction with small antigens (individual glycoproteids, their small complexes). And, respectively, the PHA test was more sensitive in reaction with large antigenic complexes (whole virions, their conglomerations, immune complexes).     

Are Patients with Erythema Migrans Who Have Leukopenia and/or Thrombocytopenia Coinfected with Anaplasma phagocytophilum or Tick-Borne Encephalitis Virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.     

Latent persistence of specific antigens of the causative agent in the composition of circulating immune complexes in typhoid fever

This study revealed the presence of O-antigen of group D salmonellae and Vi-antigen in circulating immune complexes in patients with typhoid fever, bacteriologically confirmed (56 +/- 5.6% and 65 +/- 5.4% of cases, respectively) and not confirmed (15.5 +/- 5% and 39 +/- 7% of cases, respectively), in patients with diarrhea of nontyphoid etiology in the presence of negative results of the coagglutination test and in healthy persons. The level and dynamics of circulating immune complexes were established with respect to the antigens contained in these complexes. Altogether O- and Vi-antigens were detected in circulating immune complexes, respectively, in 92%, 96% and 94% of cases on weeks 1, 2 and 3 from the beginning of the disease, and in all cases on weeks 4 and 5, the antigens being determined together and separately. Thus, the latent persistence of S. typhi antigens as part of circulating immune complexes in the blood serum was established. The determination of such persistence is of great pathogenetic and diagnostic importance, which also applies to the early period of the disease. The use of such specific and sensitive method as the coagglutination test for this purpose accelerates and facilitates the diagnosis of typhoid fever.     

Seroepidemiology of enteric fever and brucellosis in a developing country

The seroepidemiology of infection due to S. typhi and Brucella spp. in a sub-tropical, developing country of the Middle East was investigated in 130 children of age less than 1 to 15 years, 117 young adults (16-30 years) and in a further 40 adults (greater than 30 years) using an agglutination test to detect antibodies to of S. typhi, B. abortus and B. melitensis. Only 4.5% of the children's and adult's sera respectively showed the presence of antibodies to both O and H antigens of S. typhi with reciprocal titers greater than or equal to 80. Prevalence rates of 11.6%, 11.3% respectively in children and adults of antibodies to B. abortus with reciprocal titers greater than or equal to 80 and similarly 13.1%, 12.4% to B. melitensis were found. The usefulness of a single Widal test to diagnose enteric fever in a non-endemic area and the agglutination test in the diagnosis of brucellosis in an endemic area was investigated. Of 244 patients with suspected enteric fever, the sera of 100 showed reciprocal antibody titers 80 to both O and H antigens and included in these were 8 patients with concurrent culture positivity. Among 138 children and adults with suspected brucellosis, 29 culture proven cases where serologically confirmed and a further 90 were detected serologically. The sera of all the culture proven cases exhibited antibody reciprocal titer greater than or equal to 640. A similar antibody response was noted in seventy-two of the culture negative cases diagnosed serologically for brucellosis. In culture proven, cases B. melitensis was the isolate. Comparative evaluation indicated the potential usefulness of the Rose Bengal Antigen Slide Agglutination Test (Brucelloside Test) as a screening test in the serodiagnosis of human brucellosis.     

Relationships between the content of circulating immune complexes, the concentration of serum immunoglobulins and the titers of specific antibodies in patients with tick-borne encephalitis

In the blood serum of tick-borne encephalitis (TBE) patients the detection rate and concentration of circulating immune complexes, as well as the content of serum IgA, IgM and IgG, were evaluated. The formation of immune complexes was found to depend on IgM and IgG specific antibodies to TBE virus, the period of the disease and the clinical form of virus infection.     

Characteristics of Lipids Imbalance in Patients with Tick‐Borne Encephalitis

Using a novel approach, we have analyzed 30 parameters characterizing detailed spectrum and fractional content of LPs in plasma of patients with tick‐borne encephalitis (TBE). The blood plasma of all TBE patients (30 patients), as compared with that of healthy individuals (120 patients), is characterized by decreased concentrations of many LP subfractions and of the total concentration of all plasma LPs (hypolipoproteinemia). The observed difference in some parameters was statistically significant. Using computer‐assisted factor analysis, we have shown that according to these 30 parameters TBE patients are similar to patients with multiple sclerosis and systemic lupus erythematosus. The results provide grounds for using data on blood plasma LPs as additional criteria for diagnosis of TBE.     

Characteristics of lipids imbalance in patients with tick-borne encephalitis

Using a novel approach, we have analyzed 30 parameters characterizing detailed spectrum and fractional content of LPs in plasma of patients with tick-borne encephalitis (TBE). The blood plasma of all TBE patients (30 patients), as compared with that of healthy individuals (120 patients), is characterized by decreased concentrations of many LP subfractions and of the total concentration of all plasma LPs (hypolipoproteinemia). The observed difference in some parameters was statistically significant. Using computer-assisted factor analysis, we have shown that according to these 30 parameters TBE patients are similar to patients with multiple sclerosis and systemic lupus erythematosus. The results provide grounds for using data on blood plasma LPs as additional criteria for diagnosis of TBE.     

森林脑炎中和抗体蚀斑减少试验方法的建立及疫苗免疫人体后抗体检测

为评价森林脑炎疫苗的免疫效果,采用森林脑炎病毒的BHK细胞适应株,以BHK细胞为培养基质,建立了检测森林脑炎中和抗体的蚀斑减少试验方法,并用蚀斑减少法、小鼠法及ELISA法检测了原制森林脑炎灭活疫苗免疫人体后的中和抗体水平;免疫血清为按疫苗免疫程序免疫健康志愿者采血分离而制备。结果表明蚀斑减少法与小鼠法的特异性相近且敏感性较好、简便快速,缩短检测周期,为疫苗流行病学调查及新型疫苗的研究提供了快速的检测方法;原制灭活疫苗人体二针免疫后,人体血清中和抗体阳转率仅30％左右,急需提高现用原制灭活疫苗的质量。     

森林脑炎自然疫源地样本的监测及病毒的分离研究

为了解森林脑炎疫源地的分布变化趋势及样本分离病毒的特性,采集了森林脑炎高发区周边的森林全沟硬蜱、血蜱样本及森林脑炎患者的脑组织样本,用小白鼠脑内接种法检测、分离病毒分离的病毒经鉴别试验证明为森林脑炎病毒：蜱、脑两种标本检测的阳性率分别为50％和100％、结果表明森林脑炎的疫区有从林区向农业区扩散的趋势,且全沟硬蜱的带毒率较高;森脑患者的脑组织样本与蜱标本病毒的性状育差异     

Circulating immune complexes in cervical cancer patients as detected by C1q binding

In a retrospective study in women with cervical cancer, circulating immune complex levels were measured by radioimmunoprecipitation with 125I-C1q. Sera from 46 patients with cervical cancer and 35 normal controls were examined. Significantly higher levels of immune complexes were detected in cancer patients compared with controls. Mean value of binding capacity in patients was 49.8%, and by contrast, in the controls was 27.4% (two-tail test = 0). Increases in tumor mass were associated with high levels of circulating immune complexes. The presence of immune complexes in circulation statistically correlated with disease activity, however, the assay used still had limited value for diagnosis or aiding in therapeutic decisions. Nevertheless, the future holds promise for such uses.     

The Vancouver Lymphadenopathy-AIDS Study: 5. Antecedent behavioural,clinical and laboratory findings in patients with AIDS and HIV-seropositive controls.

In a group of homosexual men in Vancouver studied prospectively since November 1982, 26 cases of acquired immune deficiency syndrome (AIDS) have arisen. To identify behavioural, clinical and laboratory findings that might predict the development of AIDS in people with antibody to human immunodeficiency virus (HIV), we compared data for 25 patients with AIDS with corresponding data for 80 controls serologically positive for HIV selected from the cohort. The clinical and laboratory data for the patients with AIDS preceded the diagnosis of the syndrome by a mean of 17.5 months. The controls had been both seropositive and AIDS-free for a mean of 16.7 months after acquisition of their data. We detected significant differences between the patients with AIDS and the controls in IgG and IgA levels, absolute number of helper T cells and ratio of helper to suppressor T cells but not in lifetime number of male sexual partners, frequency of receptive anal intercourse or receptive fisting, illicit drug use or history of infectious disease. We also detected an increased risk of AIDS among those who had an elevated number of sexual contacts in AIDS-endemic areas in the 5 years before enrollment. A history of increased early sexual contact in AIDS-endemic areas is likely to be associated with early infection and with an increased risk of AIDS among men with HIV infection of unknown duration. Thus, although our analysis had limited statistical power, we conclude that most lifestyle variables appear to act as exposure factors in HIV infection but not as cofactors in the development of AIDS.     

Tick-borne encephalitis virus prevalence in Ixodes ricinus ticks collected in high risk habitats of the south-Bohemian region of the Czech Republic

A survey for tick-borne encephalitis virus in Ixodes ricinus ticks was conducted in May 2000 in two districts of the South-Bohemian region of the Czech Republic with a high occurrence of tick-borne encephalitis (TBE) in humans. Homogenized ticks were tested on PS cells, which were examined for any cytopathic effect, plaque assay and indirect fluorescent antibody test (IFT). The IFT has proved to be the most sensitive and most rapid test to demonstrate the virus in ticks. TBE virus was found in 17 of 187 pooled samples, consisting of a total of 2,968 ticks. The mean minimum infection rate was 0.6% for all tick stages combined. Infection rates in nymphs collected indifferent locations varied between 0.2 and 1.3% and between 5.9 and 11.1% in adult ticks. The observed TBE prevalence in ticks was compared with data obtained elsewhere in the Czech Republic. It is concluded that screening of ticks for TBE virus prevalence using IFT is a valuable indicator for the degree of risk to contract TBE in as particular habitat. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Basic Analysis of the Cerebrospinal Fluid: An Important Framework for Laboratory Diagnostics of the Impairment of the Central Nervous System

Laboratory analysis of basic cerebrospinal fluid (CSF) parameters is considered as essential for any CSF evaluation. It can provide rapidly very valuable information about the status of the central nervous system (CNS). Our retrospective study evaluated parameters of basic CSF analysis in cases of either infectious or non-infectious CNS involvement. Neutrophils are effector cells of innate immunity. Predominance of neutrophils was found in 98.2% of patients with purulent inflammation in CNS. Lymphocytes are cellular substrate of adaptive immunity. We found their predominance in 94.8% of patients with multiple sclerosis (MS), 66.7% of patients with tick-borne encephalitis (TBE), 92.2% of patients with neuroborreliosis, 83.3% of patients with inflammatory response with oxidative burst of macrophages in CNS and 75.0% of patients with malignant infiltration of meninges (MIM). The simultaneous assessment of aerobic and anaerobic metabolism in CSF using the coefficient of energy balance (KEB) allows us to specify the type of inflammation in CNS. We found predominantly aerobic metabolism (KEB > 28.0) in 100.0% CSF of patients with normal CSF findings and in 92.8% CSF of patients with MS. Predominant faintly anaerobic metabolism (28.0 > KEB > 20.0) in CSF was found in 71.8% patients with TBE and in 64.7% patients with neuroborreliosis. Strong anaerobic metabolism (KEB < 10.0) was found in the CSF of 99.1% patients with purulent inflammation, 100.0% patients with inflammatory response with oxidative burst of macrophages and in 80.6% patients with MIM. Joint evaluation of basic CSF parameters provides sufficient information about the immune response in the CSF compartment for rapid and reliable diagnosis of CNS involvement.     

Antibodies to DNA in the Blood of Patients with Tick-Borne Encephalitis

The relative levels of autoantibodies to native and denatured DNA (nDNA and dDNA, respectively) in the blood were compared in 55 patients with tick-borne encephalitis (TBE). Compared to healthy donors, the titers of antibodies (Ab) to nDNA and dDNA were significantly higher in 31% and 40% of TBE patients, respectively. The proportion of patients with an increased concentration of anti-nDNA Ab in the case of TBE (32%) was higher than among patients with multiple sclerosis (18%) and some other autoimmune diseases (6–18%) but comparable with that among patients with systemic lupus erythematosus (38%) and polymyositis (42%). In contrast to the patients with systemic lupus erythematosus and multiple sclerosis, the level of antibodies to nDNA in TBE patients was higher than that of anti-dDNA Ab. The coefficients of correlation between the levels of Ab to nDNA and dDNA were estimated for the entire group of TBE patients and for subgroups with different forms of the disease (temperature reactions, febrile form, and meningeal form). Analysis of correlation between the anti-DNA antibody titers and three standard biochemical markers of TBE (aspartate aminotransferase and alanine aminotransferase activities and total bilirubin) was carried out for 22 patients. A statistically significant correlation was revealed only between the level of Ab to nDNA and the marker enzyme activities, with the respective correlation coefficients being +0.44 and +0.48, respectively.     

Antibodies to DNA in the blood of patients with tick-borne encephalitis

A comparison of relative levels of autoantibodies (Abs) to both native (n) and denatured (d) DNA in the blood of 55 patients with tick-borne encephalitis (TBE) was carried out. 31% of patients with TBE was shown to have an increased level Abs to nDNA and 40% of patients demonstrate increased level of Abs to dDNA. The percent of TBE patients with increased concentration of anti-nDNA Abs higher then that in patients with multiple sclerosis (18%) and some other autoimmune diseases (6-18%), but comparable with that for patients with systemic lupus erythematosus (38%) and polymyositis (42%). In contrast to patients with systemic lupus erythematosus and multiple sclerosis, the level of Abs to nDNA in patients with TBE is higher than the level of Abs to dDNA. Correlation coefficients of Ab levels to both n- and dDNA were estimated for group of patients in whole and for separate subgroups with different type of disease (temperature reaction, feverishness and meningitis). Analysis of correlation between titres of anti-DNA Abs and three standard biochemical markers of TBE (activity of aspartate- and alanine-aminotransferases, and concentration of whole bilirubin) for 22 patients with TBE was carried out. Statistically significant correlation was revealed only between the level of Abs to nDNA and activities of aspartate- and alanine-aminotransferases, correlation coefficients are equal to +0.44 and +0.48, respectively.     

Blocking action of the soluble H-2 antigens and H-2 antibody isolated by various methods and mouse immune lymphocytes]

The treatment of tumour and lymphoid cells of mice with 3M KC1 solution having high ionic strength, nonionic detergent and by subsequent freeze-thawing resulted in obtaining serologically active H-2 antigen preparation capable of specifically blocking the cytotoxicity of H-2 antisera. The antigenic activity of the preparations thus obtained depended on the source from which they were isolated (spleen cells and their membrane fragments proved to be the best source), on the degree of maturity of tumor cells and the degree of purification of the preparation, as well as on the methods of solubilization. The blocking action of soluble H-2 antigens on the cytotoxicity of immune lymphocytes depended on the method used for isslating these antigens. The interaction of immune lymphocytes and H-2 antisera with soluble antigens was probably effected by different mechanisms.     

Circulating immune complexes and in vitro cell reactivity in paracoccidioidomycosis

The severest forms of paracoccidioidomycosis (Pcm) are associated with impaired cell-mediated immunity, a phenomenon that is reversible with therapy. It has been postulated that plasma factors could be responsible for such immune dysfunction. In this report, circulating immune complexes (CIC) were measured by the Raji cell radioimmunoassay (Raji) and by the¹²⁵I-C1q binding assay (C1q-BA) in sera from 14 patients with either active or inactive forms of Pcm and from 15 healthy controls. The C1q-B A revealed significantly elevated levels of CIC in the sera of all but one of the patients. Four of the 8 active (62%) and 2 of the 6 inactive (33%) patients had CIC levels significantly higher than the controls as determined by the Raji test. Significantly increased levels of CIC were detected only in the active patients by the Raji test. The serum of one of the patients, with a generalized infection and depressed lymphocyte responsiveness, was examined and found to contain a factor which depressed the in vitro proliferation of both homologous and normal lymphocytes. We also found that pre-culture of the patients' lymphocytes before stimulation restored their proliferative capacity, and IC were detectable in the culture supernatants. However, the subsequent addition of the patients' serum to such precultured cells did not reinduce the depression. It is suggested therefore, that the depression of T cell responses observed in Pcm is due to the presence of IC which may interact reversibly with the responding cells and/or activate a suppressor cell population whose activity is diminished by preculture.     

In vitro study of macrophage barrier function in mice vaccinated against tick-borne encephalitis and infection with flaviviruses of varying virulence

Tick-borne encephalitis (TBE) and Langat viruses were shown to be equally capable of multiplication in mouse peritoneal macrophages (PM) in vitro. The reproduction dynamics of TBE virus proved to be the same in PM of mice both highly sensitive and relatively less sensitive to TBE virus. The preliminary immunization of PM donors with commercial inactivated or experimental concentrated TBE virus vaccine produced no effect on the capacity of the virus for multiplication in PM. These facts indicate the absence of correlation between the capacity of the viruses under study for multiplication in PM in vitro and their virulence in vivo, as well as the insignificant role of circulating macrophages in the realization of the barrier function in an immune or nonimmune body.