Cytokines, autoantibodies and viral antibodies in premorbid and postdiagnostic sera from patients with rheumatoid arthritis: case-control study nested in a cohort of Norwegian blood donors.

OBJECTIVE: To assess the timing of changes in cytokines, cytokine-related markers, autoantibodies and viral antibodies in the pathogenesis of rheumatoid arthritis (RA). METHODS: Case-control study nested in a prospective cohort of 31 330 blood donors in Oslo, Norway. Forty-nine donors developed RA up to 23 years after their most recent blood donation. Stored sera from these donors (case sera) and a sex- and age-matched sample of 245 healthy donors (control sera), and postdiagnostic sera from 33 of the 49 RA cases, were analysed for a panel of cytokines and cytokine-related markers, autoantibodies and antibodies against Epstein-Barr virus and parvovirus B19. RESULTS: Cytokines and cytokine-related markers were generally negative in case sera from >5 years before the diagnosis of RA. In the 5-year interval immediately before the diagnosis of RA, more case than control sera were positive (odds ratios >2) for interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist, IL-4, IL-10, tumour necrosis factor-alpha and soluble tumour necrosis factor receptor I. In postdiagnostic sera, however, 11 of 16 examined cytokines and cytokine-related markers were statistically significantly elevated compared with control sera. Seropositivity for IgG antibodies against cyclic citrullinated peptides and for IgM and IgA rheumatoid factors were seen in case sera from up to 18 years before the diagnosis of RA. IgG antibodies against Epstein-Barr virus and parvovirus B19 did not differ significantly between case and control sera. CONCLUSIONS: Cytokines and cytokine-related markers appear to be upregulated rather late in RA pathogenesis. In contrast, IgM rheumatoid factor and IgG anti-cyclic citrullinated peptide autoantibodies may precede the diagnosis of RA by up to two decades.

A role of late Epstein-Barr virus infection in multiple sclerosis.

OBJECTIVE: To assess Epstein-Barr virus (EBV) seroconversion in a high multiple sclerosis (MS) prevalence area and to evaluate the recall of diagnosed infectious mononucleosis in MS patients. METHODS: The study was based on information or blood samples, or both, from schoolchildren, young MS patients and matched controls. EBV serology was performed on 1154 blood samples. RESULTS: We demonstrate that more than one third of the population in a high MS prevalence area is seronegative to EBV at puberty. This is in contrast to the virtually complete seroconversion to EBV early in life in individuals from areas with a low prevalence of MS. Furthermore, we demonstrate that recall of diagnosed infectious mononucleosis (IM), but not recall of common childhood diseases, is significantly more frequent among MS patients than healthy controls. All MS patients, including patients without prior immunosuppressive treatment, were EBV seropositive. CONCLUSION: During or after puberty, EBV is transmitted to a major proportion of the population in an MS high-prevalence area. Together with our previous documentation of an association between late infection with EBV and an increased risk of developing MS, these data support a role of EBV infection in MS.

Respiratory syncytial virus infection--risk factors for hospital admission: a case-control study.

UNLABELLED: Most infants are infected with respiratory syncytial virus (RSV) during the first 2 y of life. The majority have only a mild upper respiratory tract infection, but 1-2% develop a more severe illness and are admitted to hospital. AIM: To carry out a study of risk factors for hospital admission because of RSV infection in Denmark in children aged less than 2 y of age. METHODS: The study population included all 1252 children admitted to hospital with verified RSV infection in two Danish counties during the 5-y period 1990-1994. The investigation comprised a retrospective case-control study with five matched controls per case. In a multivariate analysis the risk factors included medical and demographic variables, and in infants <3 mo of age at hospitalization, two aspects of innate immunity: mannose-binding lectin (MBL) concentration and maternal RSV serum antibody titre, measured on eluates from stored dried blood from the infants' 4th day of life. The effect of each risk factor is expressed as an odds ratio, corresponding to the relative risk of being a case rather than a control if the risk factor is present. RESULTS: The following independent risk factors were identified: age, sex, month of birth, gestational age, birthweight, presence of a sibling, up to 5 y older than the case, and maternal smoking during pregnancy. There was a marginal effect of maternal RSV antibody levels, but no effect of neonatal serum MBL concentration or of crowding in the household. CONCLUSIONS: Ninety percent of cases and 80% of controls had one or more risk factors. Even though several factors were found to increase the risk for hospitalization for RSV disease, all the effects were small and no single specific factor could be identified to explain the hospitalization of the minority of children with RSV infection.

A household study of chickenpox in Guinea-Bissau: intensity of exposure is a determinant of severity.

OBJECTIVE: To determinate the relative importance of state of nutrition and intensity of exposure for clinical severity of chickenpox in a developing country. METHODS: A prospective household study was performed in a semi-urban area in Bissau, the capital of Guinea-Bissau, between December 1994 and June 1995. Antibodies were measured in the acute and the convalescence phase to assess validity of clinical diagnoses. The clinical severity of infection was assessed by number of pox, fever response and skin infections. Severity was compared for index cases, i.e. the first case in the house, and secondary and tertiary cases infected following exposure at home. RESULT: Chickenpox was diagnosed in 165 persons. The clinician's and the mothers' diagnoses corresponded well with the serological results. Median age was 36 months (range 3 months to 30.3 years). There was no correlation between nutritional status measured by arm-circumference and severity of infection. The number of pox was higher for secondary cases than for index cases (median 106 vs. 89, P<0.01), the difference being more pronounced for girls (P=0.018) than for boys (P=0.575). The risk of skin infection as a complication was correlated with the number of pox (P<0.001). CONCLUSION: Chickenpox was recognised correctly by Guinean mothers. The age distribution in Guinea-Bissau resembled the pattern in developed countries. The intensity of exposure was a major determinant of severity, especially for girls.

An epidemic of parvovirus B19 in a population of 3,596 pregnant women: a study of sociodemographic and medical risk factors.

OBJECTIVES: To estimate the incidence of human parvovirus B19 among pregnant women before and during an epidemic, to elucidate possible sociodemographic and medical risk factors during pregnancy and to estimate the association between parvovirus B19 infection and negative pregnancy outcome. DESIGN: Prospective study among pregnant women followed from their first antenatal visit before 24 full weeks of gestation until delivery. SETTING: Department of Obstetrics and Gynaecology, Odense University Hospital, Denmark, November 1992 to February 1994. METHODS: 3,596 pregnant women were invited to participate. The women were examined at first antenatal visit in the period from November 1992 to February 1994 and at delivery. The last delivery was in August 1994 and samples were thus collected before and during a large parvovirus B19 epidemic in Denmark January to September 1994. A blood sample for parvovirus B19 serology was taken at enrollment and from the umbilical cord at delivery. Three questionnaires were completed during 2nd and 3rd trimesters and a registration form at delivery. In total, 3,174 (87.6%) were enrolled and 79.5% completed the study. RESULTS: The prevalence of B19 IgG seropositivity at the first antenatal visit before 24 full weeks of gestation was 66% . The cumulative prevalence proportion of acute parvovirus B19 infection during pregnancy among IgG negative women was found to be 10.3% (IgM seropositivity and/or IgG seroconversion). The IgG seroconversion incidence increased significantly from 1.0% to 13.5% among 932 seronegative pregnant women before and during the epidemic, respectively (P < 0.001). Independent risk factors related to increased risk of B19 infection during pregnancy, adjusted for other sociodemographic and medical factors, were: children at home (adjusted OR 2.1, 95% CI 1.3-3.2); serious medical disease (adjusted OR 3.0, 95% CI 1.0-8.5); and a stressful job (adjusted OR 1.8, 95% CI 1.0-3.3). Parvovirus B 19 IgM seropositivity was associated with events of late spontaneous abortions and stillbirths (crude OR 9.9; 95% CI 3.3-29.4). CONCLUSION: Before and during an epidemic of acute B19 infection incidences were measured among pregnant women to be 1.0% and 13.5%, respectively. Three factors, significantly increasing the risk of acute B19, were identified as: having children at home; suffering from serious medical diseases; and having a stressful job. IgM positivity for parvovirus B19 was associated with negative outcome of pregnancy.

The 1994 human parvovirus B19 epidemic in Denmark: diagnostic and epidemiological experience.

In 1994 the first human parvovirus B19 (B19) epidemic to be documented in Denmark was recorded from February 2 to September 30. In total, 10,333 serum samples were tested for specific B19 IgM and IgG antibodies, using IDEIA Parvovirus B19 IgM and IgG kits. The prevalence of B19 IgM positivity was 11% for the whole period and 29% at the peak of the epidemic in week 14, declining from week 39 and onwards to 1-3%. The prevalence of B19 IgG (IgM-negative samples) was 60%, indicating an earlier infection, and the same for men and women. The gender distribution of tested patients was the same at the beginning of the epidemic as at the end of the epidemic and a year after its peak, i.e. 86% of samples were from women and only 14% from men. Age distribution for women was the same for the three periods (median age 34 years). For men the median age was 32 years, 39 years and 31 years, respectively. Only a few samples from children were tested. No change in test pattern was observed during the three periods. Approximately 75% of all samples tested were from women of childbearing age (18-45 years old), suggesting a fear of fetal complications in an actual or future pregnancy, rather than a serological verification of clinical symptoms. From the sparse clinical information that accompanied the serum sample we were not able to demonstrate that women were more likely than men to have a symptomatic B19 infection. With reservations we estimate that 14% of adverse pregnancy outcome is correlated with a B19 infection.

Routine diagnosis of herpes simplex virus (HSV) encephalitis by an internal DNA controlled HSV PCR and an IgG-capture assay for intrathecal synthesis of HSV antibodies.

BACKGROUND: The development of antiviral therapy increases the need for rapid, sensitive and reliable methods or combination of methods for diagnosis and monitoring herpes simplex encephalitis, HSE. OBJECTIVES: Evaluation of diagnostic performance of three successively developed HSV PCR assays when combined with a new capture ELISA for HSV intrathecal antibody production (ITT). STUDY DESIGN: During a 3.6 year period a total of 4.206 CSF and serum samples from about 4.140 hospitalized patients with a tentative diagnosis of HSE were analyzed by a new ELISA for ITT. 1.962 CSF samples were examined also by PCR. Clinical signs and symptoms and additional tests were obtained on all ITT and/or PCR positive patients. In 1993 the PCR was a double PCR. In 1994 the PCR was a single PCR with internal inhibition control. Positive samples were confirmed by a different confirmative PCR to increase the specificity. From 1995 the PCR was as in 1994 but samples were no longer divided in the serology routine laboratory. RESULTS: A total of 33 HSE cases was found (incidence 1.8 HSE per million people). All patients were treated with aciclovir. Three patients died, 9 patients had primary infection, 2 patients had HSE previously, and 2 patients relapsed. Only 11 patients recovered satisfactory. Of all 37 positive ITT 7 were unlikely positive. False positive PCR was seen in 1993 and 1994, due to sample-to-sample contamination during division of samples, but was not seen since 1995 when this procedure was changed. The test results depended on the state of the disease. Thus, the sensitivity, specificity, PPV and NPV for ITT were highest when performed more than 1 week after debut of symptoms whereas these values were highest using PCR within the first week. CONCLUSION: Routine PCR diagnosis of HSE type 1 and 2 is a highly sensitive and specific method that should be performed together with serological ITT to cover the whole time span from debut of symptoms to several weeks after hospitalization.

Herpes simplex virus and human papillomavirus in a population-based case-control study of cervical intraepithelial neoplasia grade II-III.

In order to evaluate the association between seropositivity for herpes simplex virus (type 1 and type 2) and cervical intraepithelial neoplacia (CIN), we analysed data from a population-based case-control study of CIN grade II-III which included Norwegian women aged 20 to 44 years, 94 cases and 228 controls. Our objectives were to determine if HSV-1 and/or HSV-2 seropositivity were independent risk factors for CIN, taking human papillomavirus exposure into account, and to elucidate the combined effect of HPV positivity and seropositivity for HSV In logistic regression analyses, the association between HSV-2 or HSV-1 seropositivity and CIN II-III was not explained by HPV (adjusted OR 3.0; 95%, CI 1.3-7.2 and adjusted OR 3.3; 95% CI 1.3-8.4, respectively). In analyses restricted to HPV-16 DNA-positive individuals, seropositivity for HSV-2 increased the risk of CIN (OR 11.1; 95% CI 1.2-105.7), whereas HSV-1 seropositivity was not significantly associated with CIN. In women positive for other HPV types, only HSV-1 seropositivity was associated with CIN (OR 8.5; 95% CI 1.3-55.8). In analyses of the HPV-16-seropositive individuals, neither HSV-1 nor HSV-2 seropositivity was associated with CIN. Compared to the reference group of jointly unexposed subjects, the HPV-16 DNA-positive women who were anti-HSV-2 negative had an increased risk of CIN (OR 29; 95% CI 12-67), whereas the risk in women who were both HPV-16 DNA-positive and HSV-2 was OR=247 (95% CI 31-1996). The estimate of interaction was strong, but did not reach significance, and our findings may suggest that the combined effect of the two viruses is of aetiological importance in cervical carcinogenesis. Furthermore, the results indicate that HPV DNA positivity is not sufficient to explain the sexual behaviour-associated risk of cervical neoplasia and that further studies on the role of genital HSV (type 1 as well as type 2) and other STDs are warranted.

Specific diagnostic methods for herpesvirus infections of the central nervous system: a consensus review by the European Union Concerted Action on Virus Meningitis and Encephalitis.

BACKGROUND: Herpesvirus infections of the central nervous system are often severe but are fortunately rare. The incidence of these infections has however, increased in recent years as a consequence of an increase in the number of immune-compromised individuals. New diagnostic procedures have improved our ability to diagnose these infections and herpesviruses may yet be implicated as the cause of further neurological diseases with no known aetiology. Methodological standards for selection and evaluation of patient materials are essential to the provision of reliable diagnosis, yet few studies have addressed this important issue. OBJECTIVES: To describe and define methodological standards and reference methodology for diagnosis of herpesvirus infections of the CNS. STUDY DESIGN: Information gathered by literature review. RESULTS: Only for herpes simplex encephalitis is there sufficient data to allow the definition of reference methodology. Good methodological standards exist but few studies have adhered to these standards. As methods for the detection of specific intrathecal antibody synthesis are well established yet under-used in diagnostic virology, the principle of these measurements is reviewed in some detail. CONCLUSIONS: Herpesvirus infections of the CNS are of increasing importance. High quality, multi-centre studies are needed to establish the value of the new diagnostic test procedures if further improvement in the diagnostic sensitivity and specificity of these procedures is to be achieved.

Assessment of the specificity of a commercial human parvovirus B19 IgM assay.

BACKGROUND: It is important to investigate a possible cross-reaction of anti-rubella IgM in the IDEIA Parvovirus B19 IgM test because many B19 infections are either asymptomatic or have clinical symptoms similar to those of rubella virus infections. Epstein-Barr virus (EBV) IgM, cytomegalovirus (CMV) IgM, measles IgM and rheumatoid factor (RF) IgM cross-reactions were also studied. OBJECTIVES: In the period from February to September 1994 (including a parvovirus B19 epidemic) more than 10,000 serum samples were examined for parvovirus B19 IgM in Denmark. This gave an opportunity to evaluate the commercial IDEIA Parvovirus B19 ELISA kit (DAKO A/S, Glostrup, Denmark), which was used routinely at Statens Serum Institut from the beginning of 1994 and onwards. STUDY DESIGN: A total of 123 parvovirus B19 IgM positive sera were tested for reactivity in rubella IgM EIA. A total of 78 rubella IgM positive sera, 60 EBV VCA-IgM positive sera, 30 CMV IgM positive sera and 24 measles virus IgM positive sera were tested for reaction in IDEIA Parvovirus B19 IgM test. Finally, 25 parvovirus IgM positive sera were tested for specific IgM against measles virus, EBV (VCA), CMV and for RF. RESULTS: One anti-B19 IgM positive serum sample reacted positively in the rubella IgM test. Of rubella IgM positive serum samples 4% cross-reacted in IDEIA Parvovirus B19 IgM test, as did 17 and 20% of EBV VCA-IgM and CMV IgM positive serum samples respectively. None of measles virus IgM positive serum samples cross-reacted in the IDEIA Parvovirus B19 IgM test. Of 25 initially parvovirus B19 IgM positive sera 20% cross-reacted in EBV VCA IgM test and 8% in the CMV IgM test. None reacted positively in measles virus IgM test; 28% showed weak reactivity in RF IgM test. CONCLUSIONS: Precautions must be taken when results of IgM assays are interpreted. Epidemiological and clinical observations must be considered.

Human herpesvirus-6 immunoglobulin G antibodies in patients with multiple sclerosis.

OBJECTIVE: To further investigate a possible correlation between human herpesvirus-6 (HHV-6) infection and multiple sclerosis by analyzing the level of HHV-6 antibodies in MS patients and healthy controls. MATERIALS AND METHODS: A total of 189 serum samples from patients with multiple sclerosis (MS) in different disease stages and 190 serum samples from healthy controls matched for age and sex were analyzed for HHV-6 antibodies using a competitive ELISA. RESULTS: There was no difference between HHV-6 IgG titers in MS patients and controls. Two of the controls were seronegative for HHV-6 versus to none of the MS-patients. There was no apparent difference in HHV-6 titers from patients in different disease stages. CONCLUSION: This study cannot support the theory that HHV-6 is a contributing factor to the development of MS, although a seroprevalence study like this would not disclose whether a late primary infection (in puberty) with HHV-6 might affect the development of MS.

Synthesis and potent anti-HIV-1 activity of novel 6-benzyluracil analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine.

Ethyl 2-alkyl-4-aryl-3-oxobutyrates were synthesized from the corresponding arylacetonitriles and 2-bromo esters. Condensation of the butyrates with thiourea followed by treatment with chloroacetic acid afforded the 5-alkyl-6-(arylmethyl)uracils. Condensation of the uracils with acetals using trimethylsilyl triflate (TMS triflate) as a catalyst gave acyclic 5-alkyl-6-(arylmethyl)uracil derivatives. 6-Benzyl-5-ethyluracil was also condensed with methyl 5-O-(tert-butyldiphenylsilyl)-2-deoxy-3-O-(phenoxythiocarbonyl+ ++)-alpha,beta-D-erythro-pentofuranoside, followed by Barton reduction and deprotection, to give the anomers of 6-benzyl-5-ethyl-2',3'-dideoxyuridine. Alkylation of the uracils with alkyl chloromethyl sulfides gave new thio analogues of HEPT. All new N1-substituted uracils were tested for activity against HIV-1, and the thio analogues were found extremely potent.

Competitive ELISA for detection of HHV-6 antibody: seroprevalence in a danish population.

A competitive HHV-6 enzyme-linked immunosorbent assay (ELISA) was established, utilizing HHV-6 rabbit polyclonal antibodies and HHV-6 cellular antigen. In total, 285 sera from Danish children aged 1-6 years were screened for HHV-6 antibodies. The lowest seropositive rate, 78% was found in the youngest age group from 1 year to 15 months. All 120 (100%) children aged 3 years and above were seropositive. The ELISA was compared with indirect immunofluorescence assay (IFA) using a group of 83 serum samples from 65 Danish patients (aged 4 weeks to 72 years). All but one of the sera (100% of patients) were positive in the ELISA, but only 71% of sera (65% of patients) were positive with both assays. The ELISA detected one seroconversion in a baby, which was not detected by IFA. The ELISA was found to be more sensitive than IFA.

[Survival of patients with zidovudine resistant HIV]. / Patientoverlevelse ved zidovudinresistent HIV.

The survival of 35 patients with AIDS or advanced HIV infection on zidovudine treatment was related to the viral sensitivity to the drug and to the CD4+ cell count. Fourteen patients died, the survivors were followed up for an average of 804 days. In a univariate Cox model, survival was strongly related to log IC90 (p = 0.0003) and to the CD4+ count (p = 0.0002). In a bivariate model, log IC90 and the CD4+ count contributed to the prediction of survival (p = 0.12 and 0.06, respectively). Large studies of combination or alternation therapy with several anti-HIV-drugs should be given high priority.

Treatment of active distal ulcerative colitis with immunoglobulin G enemas.

Seven patients with active distal ulcerative colitis were treated with IgG enemas given as a daily bedtime retention enema for two weeks. Evaluation of effect was assessed by means of sigmoidoscopy with biopsy, measuring acute phase reactants in peripheral blood, and measuring the faecal protein loss. Clinical signs of active disease were registered by the patients on a diary chart. Five patients completed the treatment period, two patients were withdrawn after 7 and 10 days due to deterioration of disease. Four patients did not register any effect, whereas one patient improved clinically. In conclusion, rectally administered IgG did not exert any effect on rectal ulcerative colitis in our study.

Varicella-zoster virus DNA in recurrent aphthous ulcers.

The present study was undertaken to examine for varicella-zoster virus (VZV) constituents in recurrent aphthous ulcers (RAU). Smears and biopsies of ulcers 0-3 days old were obtained from 20 otherwise healthy minor/major RAU patients and from two patients with Behçet's syndrome. VZV could not be cultivated from any of the biopsies, all smears were negative for VZV antigen, and in situ hybridization on the five samples tested was negative for VZV nucleic acid. DNA extracted from biopsies, however, yielded positive polymerase chain reaction (PCR) products in all cases with one primer set of the protein kinase 66 (PK66). Ten control biopsies from non-RAU subjects yielded no PCR products apart from the sample from AP, who obtained the biopsies, and from HOM, who performed the PCR, suggesting that the DNA had been passed on from the patients. Other parts of the PK66 gene and of thymidine kinase 36 yielded no products. Although the consistent presence of VZV DNA in RAU might suggest a pathogenetic implication, the potential presence and possible pathogenetic importance of other herpesviruses cannot be excluded.

Synthesis of pyranoid analogues of the anti-HIV active 3'-deoxy-2',3'-didehydrothymidine (D4T).

The primary hydroxy group of ethyl 2,3-dideoxy-alpha-D-erythro-hex-2-enopyranoside (1) was selectively protected and the secondary hydroxy group was deoxygenated via the dithiocarbonate 3 from which ethyl 6-O-(4-methoxybenzoyl)-2,3,4-trideoxy-alpha-D-glycero-hex-2-eno pyranoside (4) and its regioisomer (5) were produced. These were converted into didehydro nucleosides by glycosylation of silylated heterocyclic bases in the presence of trimethylsilyl trifluoromethanesulfonate as catalyst. The configurations of the anomeric products were assigned by 1H-NMR analysis of the corresponding saturated compounds which were obtained by hydrogenation of the double bond in the carbohydrate moiety. The compounds 9a,b,d, 10a,b, 14a,b,e,f, and 15a,b,e,f did not show any significant activity against HIV or HSV-1.

Development of resistance to zidovudine in HIV strains isolated from CD4+ lymphocytes and plasma during therapy.

An assay based on production of HIV antigen in cultures of CD4+ lymphocytes infected 'in vitro' with cell-free virus was established. Using this assay it was possible to isolate, propagate and reliably determine the zidovudine susceptibility of HIV isolates from all patients despite differences in cellular tropism and syncytium inducing capacity. Using this assay, differences in zidovudine susceptibility of 52 serial isolates obtained from 16 patients before and after initiation of therapy were examined. HIV with a 10- to 100-fold reduced susceptibility to zidovudine were isolated from 13 patients as early as 4 months after initiation of therapy. Number of months of zidovudine treatment was strongly associated with development of viral resistance, and high CD4 cell counts tended to be associated with lower rates of development of resistance. That patients can harbor mixtures of virus strains with different susceptibility to zidovudine was confirmed by the differences in susceptibility between isolates obtained simultaneously from CD4+ lymphocyte and plasma, and by the differences in susceptibility between virus strains isolated from clones of CD4+ lymphocytes.