Seroepidemiological studies indicate frequent and repeated exposure to Campylobacter spp. during childhood.

The annual number of episodes of clinical gastroenteritis caused by Campylobacter spp. in The Netherlands is estimated to be 75 000, i.e. once per 200 person life-years. This number is based on extrapolation of culture results from population-based studies. The number of culture-confirmed cases of Campylobacter infection peaks in the first 3 years of life and again between the ages of 20 and 25 years. The seroepidemiology of Campylobacter describes the relationship between age and exposure to Campylobacter and reflects both symptomatic and asymptomatic infections. Using a validated ELISA system, antibodies to Campylobacter were measured in an age-stratified sample (n=456) of the PIENTER serum collection of the Dutch general population. The seroprevalence of Campylobacter IgG antibodies increased with age, reaching almost 100% at age 20 years. Antibody levels steadily increased with age until young adulthood, suggesting repeated exposure to Campylobacter. In conclusion, seroepidemiological data demonstrated repeated exposures to Campylobacter throughout life, most of which do not lead to clinical symptoms. From young adulthood, >95% of the population in The Netherlands had serological evidence for exposure to Campylobacter.

Validation of an ELISA for the diagnosis of recent Campylobacter infections in Guillain-Barré and reactive arthritis patients.

Weeks or months following Campylobacter infection, a small proportion of infected individuals develop Guillain-Barré syndrome (GBS) or reactive arthritis (ReA). Stool culture for Campylobacter is often negative in these patients, and serology is therefore the method of choice for diagnosing a recent infection with Campylobacter. This study developed a capture ELISA system to detect anti-Campylobacter IgA and IgM antibodies indicative of a recent infection. The sensitivity of the assay was 82.0% in uncomplicated Campylobacter enteritis patients, 96.2% in GBS patients who were culture-positive for Campylobacter, and 93.1% in culture-positive ReA patients, with a specificity of 93.0%. The assay allows identification of Campylobacter infection in patients with post-infectious neurological and rheumatological complications.

Detection of antibodies cross-reactive with type C RNA tumor viral p30 protein in human sera and exudate fluids.

Human sera and human exudate fluids were surveyed for the presence of antibodies cross-reacting with a type C viral protein with a molecular weight of 30,000 (p30). Antibodies cross-reactive with type C viral p30 were concentrated by means of affinity chromatography using Sepharose beads to which disrupted Simian sarcoma virus or Rauscher murine leukemia virus p30 had been coupled. Eluates obtained from the affinity beads were tested for activity against Rauscher murine leukemia virus p30, Simian sarcoma virus p30, and endogenous feline virus RD-114 p30 by radioimmunoprecipitation. Of 57 eluates tested, 29 showed activity against type C viral p30. A number of eluates were also tested for anti-p30 activity by a visual microcytotoxicity assay. Of 11 eluates positive in the radioimmunoprecipitation assay, five were also positive in the microcytotoxicity assay.

The spectrum of antecedent infections in Guillain-Barré syndrome: a case-control study.

OBJECTIVE: To determine which antecedent infections are specifically associated with the Guillain-Barré syndrome (GBS). BACKGROUND: Infections with many agents have been reported preceding GBS. Some infections are related to specific clinical and immunologic subgroups in GBS. Most agents were reported in case reports and uncontrolled small series of GBS patients only, and their relation to GBS and its subgroups remains unclear. METHOD: A serologic study for 16 infectious agents in 154 GBS patients and 154 sex- and age-matched controls with other neurologic diseases. Acute phase, pretreatment samples were used from clinically well-defined GBS patients. The seasonal distribution of serum sampling in the GBS and control group was the same. RESULTS: Multivariate analysis showed that in GBS patients, infections with Campylobacter jejuni (32%), cytomegalovirus (13%), and Epstein-Barr virus (10%) were significantly more frequent than in controls. Mycoplasma pneumoniae infections occurred more often in GBS patients (5%) than in controls in univariate analysis. Infections with Haemophilus influenzae (1%), parainfluenza 1 virus (1%), influenza A virus (1%), influenza B virus (1%), adenovirus (1%), herpes simplex virus (1%), and varicella zoster virus (1%) were also demonstrated in GBS patients, but not more frequently than in controls. C. jejuni infections were associated with antibodies to the gangliosides GM1 and GD1b and with a severe pure motor form of GBS. Cytomegalovirus infections were associated with antibodies to the ganglioside GM2 and with severe motor sensory deficits. Other infections were not related to specific antiganglioside antibodies and neurologic patterns. CONCLUSIONS: Recent infections with C. jejuni, cytomegalovirus, Epstein-Barr virus, and M. pneumoniae are specifically related to GBS. The variety of infections may contribute to the clinical and immunologic heterogeneity of GBS.

The antigen spot test (AST): a highly sensitive assay for the detection of antibodies.

A method is described for detection of antibodies by means of nitrocellulose or diazobenzyloxymethyl (DBM) paper on which various antigens have been spotted. The sensitivity of this antigen spot test (AST) is comparable with that of RIA and ELISA. The method requires only nanogram amounts of antigen. Since a variety of antigens can be spotted on a single piece of nitrocellulose or DBM paper, this antigen spot test is especially useful for specificity controls on antibodies.

Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays.

The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.

Cross-reactive anti-galactocerebroside antibodies and Mycoplasma pneumoniae infections in Guillain-Barré syndrome.

Anti-galactocerebroside (GalC) antibodies are reported to be present in GBS patients with preceding Mycoplasma pneumoniae (MP) infection. We investigated the presence of anti-GalC reactivity in serum of a large group of GBS patients using ELISA and compared this with healthy controls and individuals with an uncomplicated MP infection. Anti-GalC antibody reactivity was present in 12% of the GBS patients. Furthermore, anti-GalC antibodies were associated with MP infections, a relatively mild form of the disease and demyelinating features. Anti-GalC antibodies cross-reacted with MP antigen. In conclusion, anti-GalC antibodies in GBS patients may be induced by molecular mimicry with MP.

Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer.

The prognostic value of detection of human papillomavirus (HPV) type 16 DNA in histologically cancer free lymph nodes was assessed in left obturator lymph nodes from cervical cancer patients with HPV-16 positive primary tumours. HPV-16 DNA was detected by polymerase chain reaction in 12 of 35 patients with histologically cancer free lymph nodes. Of these 12 patients, only one developed a recurrence, suggesting HPV-16 DNA detection in cancer free lymph nodes has no prognostic value.

Antibodies to human papillomavirus type 16 E7 related to clinicopathological data in patients with cervical carcinoma.

AIMS: To investigate the correlation between antibodies to the transforming protein E7 of human papillomavirus (HPV) type 16 and clinicopathological indices in women with cervical squamous carcinoma. METHODS: A synthetic peptide of the HPV type 16 E7 protein (amino acids 6 to 35) was used to screen sera from 29 children, 130 women with cervical intraepithelial neoplasia, 443 women with cervical cancer, and 222 controls, for antibodies against this viral antigen. Bivariate and multivariate analyses were used to investigate the correlation between the serological status in the pretreatment sera and clinicopathological indices (size of the lesions, histological grade, stomal infiltration, vascular invasion, and nodal spread). Survival analysis was done using the Cox regression model for all FIGO stages and stages IB and ILA. RESULTS: Cervical carcinoma patients had a significantly higher prevalence of antibodies to synthetic peptide E7/6-35 than women with cervical intraepithelial neoplasia (17.7% v 7%, p < 0.005) or controls (17.7% v 11%, p < 0.05). Bivariate analysis of the data on the presence of anti-E7/6-35 antibodies in the pretreatment sera from these patients and clinicopathological indices showed a significant correlation between the presence of anti-E7/6-35 antibodies and the size of the lesion (p = 0.0009), histological grade (p = 0.0031), and lymph node metastasis (p = 0.01). 0.011). In addition, the Cox regression model, analysing four risk factors which can be determined before treatment, showed a significant correlation between the presence of anti-E7/6-35 antibodies and a worse prognosis (p = 0.003). Survival analysis revealed that both for all FIGO stages (p = 0.0005) and for stages IB and IIA alone (p = 0.0021), anti-E7/6-35 positive patients before treatment had a significantly shorter life expectancy. CONCLUSIONS: The presence of antibodies against E7/6-35 in pretreatment sera from patients with cervical carcinoma correlates with the size of the lesions, lymph node involvement, and a worse prognosis.

Detection of antibodies against Legionella pneumophila serogroups 1 to 6 and the Leiden-1 strain by micro ELISA and immunofluorescence assay.

One hundred and seventy-five human sera were tested for the presence of type-specific antibodies against L pneumophila serogroups 1 to 6 and the Leiden-1 strain by means of an enzyme linked immunosorbent assay (ELISA) and compared with the results obtained by the indirect immunofluorescence assay (IFA). A high correlation (correlation coefficient 0.92) between both methods was found. No consistent pattern of IgG, IgA and IgM classes of antibody titres against L pneumophila were found. In the sera of 15 of 17 patients with a proven L pneumophila pneumonia, IgM class antibodies against L pneumophila could be detected. A "polyvalent" ELISA was developed which permits rapid routine screening of human sera for antibodies against L pneumophila serogroups 1 to 6 and the Leiden-1 strain.

Epstein-Barr virus specific marker molecules for early diagnosis of infectious mononucleosis.

The molecular specificity of the antibody response against Epstein-Barr Virus (EBV) is studied in patients with acute primary EBV-infection, i.e. infectious mononucleosis syndrome. Using the immunoblot technique both IgM and IgG antibody responses are studied in sera obtained serially until week 20 after onset of symptoms. Healthy seropositive blood donors are used as control. Antigens are prepared from virus producer cell lines B95-8, P3HR1 and HH514-c16 (a superinducible derivative of P3HR1), induced for the expression of early antigens (EA) or viral capsid antigens (VCA) and from the EBV-negative cell lines BJAB and RAMOS. The 'WC'-serum, described by Edson et al. (J. Immunol. 130, 1983) is used to characterize EA- and VCA-specific polypeptides and to define their subcellular location. The immunoblot studies reveal an enormous diversity in EBV-specific polypeptides recognised by different individual patients, both for IgM and IgG. In addition, these patterns were markedly different from those found with control blood donor sera. The latter predominantly recognised bands at 72 kDa (EBNA) and 41 and 18 kDa respectively (both VCA components). Despite the great individual variation observed, EA-specific polypeptides at 138 kDa and at 45-52 kDa were recognised by both IgM and IgG antibodies in first serum samples of all patients tested.

Comparison of in situ DNA hybridization and immunological staining with conventional virus isolation for the detection of human cytomegalovirus infection in cell cultures.

The sensitivity of immunochemical staining and in situ DNA hybridization for the detection of human cytomegalovirus (HCMV) was compared with that of virus isolation. Human diploid fibroblasts were infected with serial, 10-fold dilutions of HCMV strain AD169 and examined at various intervals between 1 and 42 days after inoculation, using the three methods being compared. HCMV-DNA was detected by in situ hybridization using a biotin-labeled HCMV probe and CMV early antigen (EA) by immunochemistry using a specific monoclonal antibody. During the first 2 days after inoculation detection of EA appeared to be the most sensitive method. After the fifth day the sensitivity of the immunochemical and in situ hybridization methods was similar and equalled that of conventional virus isolation. However, 2-5 times more HCMV-DNA than HCMV-EA positive cells were detected. Our results indicate that both detection of HCMV-EA by immunological staining and HCMV-DNA by in situ hybridization are suitable methods for rapid and sensitive detection of HCMV infections.

Natural antibodies in human sera cross-reactive with type C RNA tumor viral P 30.

Antibodies cross-reactive with type C viral p30 were concentrated from 105 samples of human plasma by affinity chromatography using Sepharose beads to which disrupted Simian sarcoma associated virus (SSAV) had been coupled. Eluates obtained from the affinity beads were tested for anti-p30 activity by performing radio-immunoprecipitations with 125I-labeled SSAV p30 and Rauscher murine leukemia viral (R-MuLV) p30. Forty six percent of the eluates were found positive when tested against SSAV p30. From 44 positive eluates for anti SSAV p30 activity, 20 (45%) were also found to be positive for anti-R-MuLV p30 activity. No significant difference was found in anti-p30 activity in eluates from normal controls and from patients with various kinds of malignancy.

Prediction and diagnosis of type 1 diabetes using beta-cell autoantibodies.

The clinical manifestation of type 1 diabetes is the endpoint of a long-lasting immune-mediated destruction process of the B-cells. Autoantibodies originating from this process can be applied in the diagnosis and clinical discrimination of autoimmune diabetes as well as in the prediction of this disease. At clinical diagnosis between 80-90% of patients with type 1 diabetes are positive for antibodies to B-cell antigens, such as ICA and antibodies to glutamic acid decarboxylase or IA2. These antibodies can also be detected in the presymptomatic period before onset of the disease, and can thus be used to predict type 1 diabetes. Using a combination of antibodies, diabetes can be predicted in 70-80% of future cases of diabetes, with a positive predictive value between 30-80%, depending on the type of antibody tested for and the population studied. Between 5 and 30% of patients initially diagnosed with type 2 diabetes will show progression to insulin dependency and turn out to have type 1 within three years of diagnosis. It is clinically relevant to identify these patients early in the course of disease, as deterioration of metabolic control results in an increased risk for macro- and micro-vascular complications. Autoantibodies to glutamic acid decarboxylase or ICA are of high diagnostic sensitivity in these cases and are better predictors for future insulin dependency than biochemical or clinical parameters. Increasing knowledge on the applicability of antibodies for diabetes prediction and diagnosis and the development of commercial assays for antibodies to glutamic acid decarboxylase and IA2 antibodies has enabled the implementation of B-cell autoantibodies in routine diagnostic settings.

Cardiolipin antibodies and lupus anticoagulant in young patients with a cerebrovascular accident in the past.

To determine whether young patients who suffered a stroke in the past, have a higher prevalence of ACA of LAC as compared to healthy controls, we evaluated 44 stroke patients and 46 controls in a case-control study for the presence of ACA and LAC. All the patients had had a stroke under the age of 50 yr and the stroke date was less than 5 yr ago (mean 2.5 yr). Stroke was defined as an ischaemic cerebral infarction and was confirmed by angiography, CT-scan or MRI. An age- and sex-matched group of healthy volunteers served as controls. The mean age of the patients was 41.4 yr (range 22-52 yr), and of the controls 36.8 yr (range 24-50 yr). Serum and plasma from both groups was examined for IgM- and IgG-ACA and LAC. One patient was positive for both IgG- and IgM-ACA, whereas 3 controls were found positive for IgG-ACA. For 2 patients and 5 controls an equivocal result was obtained for IgG-ACA or IgM-ACA. None of the patients or controls were positive for LAC. The differences between the patient and control group were statistically not significant. In conclusion, no difference was found in the prevalence of cardiolipin antibodies in sera from patients with a stroke within the last 5 yr and an age- and sex-matched control group. There was no correlation either between the presence of lupus anticoagulant and the occurrence of a stroke in the past.

Gastroenteritis caused by Campylobacter concisus.

We describe a case of gastroenteritis caused by Campylobacter concisus. The pathogenic potential of C. concisus has yet to be elucidated. Recent studies indicate an association with enteric disease in immunocompromised patients and inflammatory bowel disease in children. Molecular identification methods may be necessary for identifying certain Campylobacter species because of phenotypic similarity.

Axonal variant of Guillain-Barre syndrome associated with Campylobacter infection in Bangladesh.

BACKGROUND: Campylobacter jejuni enteritis is the predominant bacterial infection preceding Guillain-Barré syndrome (GBS), an acute postinfectious immune-mediated polyradiculoneuropathy. The purpose of this study was to define the clinical phenotype of GBS and the relation with preceding C jejuni infections in Bangladesh. METHODS: We performed a prospective matched case-control hospital surveillance including 100 patients fulfilling the National Institute of Neurological Disorders and Stroke criteria for GBS from 2006 to 2007 in the Dhaka area of Bangladesh. Detailed clinical, electrophysiologic, serologic, and microbiologic data were obtained with a follow-up of 6 months. RESULTS: GBS affected predominantly young adult males living in rural areas. Sixty-nine percent of the patients had clinical evidence of a preceding infection. The most frequent symptom was diarrhea (36%). The majority of patients had a pure motor variant of GBS (92%) with relatively infrequent cranial nerve involvement (30%). Twenty-five percent of patients required respiratory support. Electrophysiologic studies showed that 67% of patients had an axonal variant of GBS. Eleven patients (14%) died, and 23 (29%) remained severely disabled during the follow-up. Positive C jejuni serology was found in an unprecedented high frequency of 57% as compared with 8% in family controls and 3% in control patients with other neurologic diseases (p < 0.001). C jejuni infection was significantly associated with serum antibodies to the gangliosides GM1 and GD1a, axonal neuropathy, and greater disability. CONCLUSIONS: We report an unusually high frequency of the axonal variant of Guillain-Barré syndrome in Bangladesh, associated with preceding Campylobacter jejuni infection, severe residual disability, and high mortality.

Measurement of cytokine antibodies. Test development.

Several assays have been used for detection of antibodies against cytokines. The choice of assay is greatly dependent on the intended goal, e.g. detection of naturally occurring antibodies or therapy induced antibodies. The different assays can be grouped in 2 categories. The interference or indirect assays are based on the detection of the test sample interference with the biological activity, with detection of the cytokine in EIA or with binding to cellular receptors. In direct assays cytokine antibodies are detected by binding to solid phase fixed cytokines, followed by incubation with a secondary enzyme-labelled anti-human Ig antibody or by binding to 125I-labelled cytokines in RIA.