Use of ELISA to measure antinuclear antibodies in children with juvenile rheumatoid arthritis.

OBJECTIVE: To compare a series of commercial ELISA tests with an indirect immunofluorescent antibody (IFA) test for the detection of antinuclear antibodies (ANA) in children with juvenile rheumatoid arthritis (JRA). METHODS: Sera from 178 patients with JRA (88 pauciarticular, 68 polyarticular, 22 systemic) were compared with 26 healthy pediatric subjects. Twenty-one samples from patients with systemic lupus erythematosus (SLE) were also tested. All samples were analyzed by IFA and by 3 commercial ELISA methods. Concordance of ELISA results with IFA results (selected standard) were used as a measure of performance. Sensitivity and specificity were calculated for each test and likelihood ratios (LR) were established for IFA and ELISA in pauciarticular and polyarticular JRA sera. The increment in pretest probability was then obtained for each test as an additional measure of test performance. RESULTS: IFA rendered positive results on 18-77% of the JRA sera depending upon the subset, 100% of SLE sera, and 15% of normal patient sera. Using IFA as the standard, correspondence with positive results among patients with JRA ranged from 0 to 74% for the 3 ELISA tests, while it ranged from 5 to 73% in IFA negative sera. IFA tests showed intermediate range likelihood ratios (0.3, 0.5, 3.5, and 5) and increments in pretest probability ranging from 25 to 45%. While one of the ELISA tests attained 50% of increment in pretest probability for the positive test, it showed 0% increment as a negative test. The other 2 ELISA tests incremented the pretest probability from 0 to 25%. CONCLUSION: Our findings indicate that in JRA, the lack of correspondence with the historic standard IFA precludes the use of ELISA tests for detection of ANA. In addition, IFA out-performs ELISA by a substantial degree when "clinical utility" analysis of test performance is utilized. Detection of ANA in children with JRA should either continue to rely on IFA or be based on a different set of antigens if an ELISA format is chosen.

Comparison of immunodot and western blot assays for diagnosing Lyme borreliosis.

Two commercially available serologic tests for use in diagnosing Lyme borreliosis were evaluated by using a test panel comprised of sera from patients diagnosed with Lyme borreliosis, non-Lyme disease controls, and healthy subjects. The test methods examined were a Western blot assay and an immunodot assay. The study was initiated to determine how the immunodot assay, which contains purified and recombinant proteins to those borrelial antigens recommended for immunoglobulin M (IgM) detection in the Dearborn criteria, would compare with the Western blot assay as a confirmatory method for serologic diagnosis of Lyme borreliosis. Results obtained showed that the two test methods performed comparably for detecting IgG antibodies. For IgM antibody detection, the immunodot and Western blot assays had similar sensitivities; however, the immunodot assay was more specific and had greater positive predictive value than the Western blot assay. The results obtained indicate that the immunodot assay performs as well as or better than the Western blot assay for diagnosing Lyme borreliosis. Furthermore, because it uses a limited panel (n = 5) of antigens, the immunodot is easier to read and interpret than standard Western blots.

Correlation of seroreactivity with response to antibiotics in pediatric Lyme borreliosis.

Response to treatment with antibiotics was compared with serologic reactivity and clinical symptoms in a pediatric population with presumptive diagnoses of Lyme borreliosis. The population analyzed for this study consisted of a subset of a larger Lyme clinic population being monitored as part of a prospective study on pediatric Lyme borreliosis. All patients resided in an area in which Ixodes scapularis and Borrelia burgdorferi are considered endemic. Serum from patients was tested by enzyme-linked immunosorbent assay and Western blotting. Response to antibiotics was evaluated by members of a pediatric Lyme clinic. Results showed that positive serologic test results correlate with a favorable response to antibiotics, as does the presence of erythema migrans (EM), regardless of serologic status. Seronegative patients without EM had chronic fatigue and arthralgia and/or myalgia as primary symptoms and did not respond to antibiotics, even when multiple courses of treatment were given. These results indicate that serologic tests designed to have high specificity can reliably rule out Lyme borreliosis in patients with chronic symptoms, thus preventing unnecessary treatment with antibiotics.

Residual serologic reactivity in children with resolved Lyme arthritis.

OBJECTIVE: To define the pattern of persistent antibody response in children with resolved Lyme arthritis. METHODS: From a cohort of 67 children with Lyme arthritis followed in our department since 1989, 19 were selected using these criteria: All patients (1) were asymptomatic; (2) had an ELISA titer < or = 1:160; (3) had been in treatment a minimum of 6 months. Their initial and late samples were assessed by Western blot and the pattern of reactivity was analyzed. RESULTS: The mean interval between treatment and last sample was 9.6 months (6-23). Analysis of the last sample showed that only 5/19 were negative by ELISA and 4/19 were at the cutoff limit (1:80). Only 6 patients had fewer than 4 reactive bands, 4 had 4 bands, and 9 had 5-11 bands on Western blot. The 41, 39, and 60 kDa were the most commonly observed reactive bands at last evaluation. 31 and 34 kDa bands, while relatively common in initial samples (36%), became uncommon (5%) on late samples. A significant finding was the absence of IgM reactivity in 18/19: 1/19 had 41 kDa reactivity. Only 4 patients had both ELISA (< 1:80) and Western blot tests negative (< 5 reactive bands). CONCLUSION: All patients with resolved Lyme arthritis continue to show serologic reactivity beyond 6 months of therapy. 68% of the patients satisfy Western blot criteria for positivity in our laboratory. IgM reactivity to any antigen was minimal and IgG reactivity against the 41 kDa antigen, considered diagnostic of infection in initial samples by some laboratories, is very common (16/19).

The overdiagnosis of Lyme disease in children residing in an endemic area.

The medical records of 227 children ages 1 to 19 years referred to the Lyme disease pediatric clinic over a 32-month period since May 1990 were reviewed. Clinico-serologic criteria for a positive diagnosis were applied. One hundred thirty-eight of 227 referred children did not fulfill those criteria and became the study population. Four subsets of patients emerged: (1) 54 patients with predominantly subjective symptoms; (2) 52 patients with objective evidence for an alternative diagnosis; (3) eight patients who had documented infection in the past and continued with symptoms after antibiotic treatment; and (4) 24 patients with a history of tick attachment or prenatal/family history of Lyme disease. Serologic testing data from commercial laboratories were available for the 54 children from the "predominantly subjective" group; 50% were negative, and 50% were borderline or positive. Ninety-two percent of these patients were negative at retesting by our enzyme-linked immunosorbent assay (ELISA) and 100% were negative by Western blot. Fifty-seven percent of these patients had received treatment prior to our evaluation. Children residing in an endemic area who present with vague symptoms are being diagnosed with and treated for Lyme disease without clinical or serologic documentation. In addition, fear in the lay community may be inducing doctors to diagnose Lyme disease in patients with symptoms that may be suggestive of an alternative diagnosis.

Serial measurements of soluble interleukin 2 receptor levels (sIL2-R) in children with juvenile rheumatoid arthritis treated with oral methotrexate.

OBJECTIVE: To investigate the potential clinical utility of serial levels of sIL2-R as a marker of disease activity among children with juvenile rheumatoid arthritis (JRA) treated with methotrexate (MTX). METHODS: sIL2-R levels, measured by ELISA, were evaluated in 16 JRA patients (10 polyarticular, six systemic-onset) treated with oral, weekly MTX. sIL2-R values were compared with those of 49 normal controls. Medical record review was used to obtain relevant clinical data. Joint counts (number of swollen joints) were used as indicators of clinical change. A reduction of 50% in joint counts between pre and post treatment measurements was considered a clinically significant response. RESULTS: The mean (SEM) sIL2-R value of pre treatment JRA of 1728(290) U/ml was significantly higher than the post treatment value of 921(229) U/ml (Wilcoxon Rank test, p < or = 0.001). Pre treatment values were also significantly different from the mean(SEM) of healthy controls of 519(19) U/ml (p < 0.001). Pre treatment sIL2-R levels of 2417(291) U/ml in systemic-onset JRA were significantly higher than sIL2-R values in polyarticular JRA patients of 1218(884) U/ml (Mann-Whitney rank test p < 0.001). Among the 13/16 children with good therapeutic responses (> or = 50% improved), the range of sIL2-R decreases was 154-2641 U/ml (mean 842 U/ml); sIL2-R levels increased in the three children with poor clinical responses to methotrexate. CONCLUSIONS: sIL2-R levels paralleled the course of disease in all patients. sIL2-R levels may be useful for monitoring therapeutic responses in children with JRA.

Pediatric Lyme arthritis: clinical spectrum and outcome.

A cohort of children with Lyme arthritis was used to evaluate the clinical and serologic profile of the disease. During a 42-month period (June 1989 to December 1991), 44 patients (13 girls and 31 boys, ages 4-18 years) were included and followed for 6-36 months. Inclusion required the presence of arthritis, as well as positive serology. Thirty-four children with juvenile rheumatoid arthritis or spondyloarthropathy were used as a serologic comparison group. Five different patterns of arthritis were found. Preceding erythema migrans was seen in seven children. Antinuclear antibodies were positive in 30% of the patients. Three treatments were used and selected according to physician preference, patient age, and presence of extraarticular disease: amoxicillin, doxycycline, and ceftriaxone. Articular disease reached complete resolution in all patients within 2-12 weeks. Lyme arthritis in children may mimic other pediatric arthritides. Prognosis for children with clearly defined Lyme arthritis was excellent.

Serial measurement of soluble interleukin 2 receptor levels: an early indicator of treatment response for Lyme disease.

Detection of antibodies produced in response to infection with Borrelia burgdorferi provides a valuable aid for diagnosing Lyme disease. However, anti-Borrelial antibody titers are of little value in determining treatment success or providing evidence of persistent infection as levels of specific antibodies can remain elevated even after successful treatment. Pretreatment and posttreatment measurement of soluble interleukin 2 receptor (sIL-2R) levels was evaluated for use in predicting treatment response in Lyme disease. Results indicate that serial measurement of serum sIL-2R levels can provide an early indicator of response to treatment and outcome.

Detection of antibodies to the recombinant P39 protein of Borrelia burgdorferi using enzyme immunoassay and immunoblotting.

The diagnostic value of serologic tests using the recombinant P39 protein of Borrelia burgdorferi was compared with that of tests prepared from a whole spirochete antigen source. Immunoassays (ELISA and Western blot) prepared from either the recombinant protein or whole spirochetes were evaluated using a test panel comprised of 2 sera groups, one obtained from patients with clinically diagnosed Lyme disease, the other from individuals with no indication of past or current infection with B. burgdorferi. Results obtained indicate that ELISA screening tests relying on the recombinant protein are less sensitive than ELISA tests using whole spirochete antigen preparations. Western blot tests based on the P39 protein were more specific than P39 ELISA yielding no false positive or indeterminate results. These findings suggest that the P39 protein may prove valuable for confirmation testing for Lyme disease.

Pharmacological management of juvenile rheumatoid arthritis.

The goals of pharmacotherapy in juvenile rheumatoid arthritis (JRA) are to suppress chronic synovitis which causes potential cartilage destruction and deformities, to control the systemic effects of inflammation (including growth retardation and nutritional deficits), relieve pain and limit psychological impact of disease. Currently available methods include nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, salicylates, naproxen, tolmetin, ibuprofen and indomethacin; disease modifying antirheumatic drugs (DMARDs) such as oral and injectable gold salts, hydroxychloroquine, penicillamine, oral and injectable methotrexate, and sulfasalazine; oral (daily or on alternate days), intravenous pulse or intra-articular corticosteroids; immunosuppresants, including cyclophosphamide, chlorambucil, cyclosporin, and azathioprine; and gammaglobulin and other experimental therapies. Over the past 10 years, rheumatologists have adopted more aggressive pharmacological treatment of JRA. As time progresses and the safety of certain drugs such as methotrexate and sulfasalazine becomes clearer, wider and earlier use of these agents can be expected. Still the approach to treatment is a 'step by step' one, starting with the classical NSAIDs and ending with the DMARDs as needed.

Frequency and specificity of antibodies that crossreact with Borrelia burgdorferi antigens.

The frequency and specificity of antibodies that bind antigens of Borrelia burgdorferi in sera from 200 individuals with no evidence of past or current Lyme disease was determined. Sera were tested for both IgG and IgM antibodies to B. burgdorferi by Western blotting. The non-Lyme serum group included specimens from healthy adults and children in addition to specimens from patients with viral infection and rheumatic diseases. Crossreactive IgG antibodies occurred more frequently than IgM antibodies. The most frequently bound antigens corresponded to 41 kDa and 60 kDa Borrelial components. Of 200 specimens tested, 100 had antibodies that bound at least 1 antigen. Binding to multiple antigens occurred at much lower frequency. Our results indicate that determination of maximum crossreactivity of non-Lyme sera can be used to establish minimum criteria for determining a positive Western blot result for Lyme disease.

Use of western blot and enzyme-linked immunosorbent assays to assist in the diagnosis of Lyme disease.

Without evidence of erythema chronicum migrans, diagnostic confirmation of Lyme disease may be difficult, particularly if there are conflicting laboratory results. Often, for families and physicians, the clinical dilemma is whether fatigue, arthritis/arthralgias, a positive enzyme-linked immunosorbent assay (ELISA), and tick exposure, but no evidence of erythema chronicum migrans, are sufficient to diagnose and treat Lyme disease. Patients with discordant ELISA and Western blot (WB) assay results for Borrelia burgdorferi were studied to determine whether there was sufficient clinical evidence to support a diagnosis of Lyme disease. Of 650 consecutive sera analyzed by ELISA in a laboratory within a 1-year period, 77 were subsequently tested by WB. The clinical data from these patients were then analyzed. The study population was divided into three groups: group 1 (positive ELISA, positive WB), group 2 (positive ELISA, negative WB), and group 3 (negative ELISA, negative WB). Findings included the following: (1) Patients with a strong clinical history of Lyme disease were usually positive by both WB and ELISA (group 1). (2) All patients with erythema chronicum migrans had both positive WB and ELISA tests. (3) Ninety-one percent of group 2 had a rheumatic or inflammatory condition other than Lyme disease. (4) A definite response to antibiotics occurred in 75% of patients wherein both ELISA and WB were positive but in only 11% of cases with a positive ELISA but a negative WB. (5) History of tick exposure and degree of fever were not significantly different among the three serologic groups, and thus they were not diagnostically helpful.(ABSTRACT TRUNCATED AT 250 WORDS)

Chief resident training. Developing leadership skills for future medical leaders.

Most future medical leaders receive little or no formal exposure to management and leadership training activities. Industry invests considerable resources in such training for middle and senior level managers. With the increasing complexity of health care delivery systems and the increased involvement of diverse teams of individuals in the care of patients, it was believed that a training activity designed to enhance participant human interaction skills and teach about group behavior and team management might be valuable to pediatric chief residents. This article describes a 3-day workshop designed to develop such leadership skills. It has been conducted for 3 years with 117 participants. Results of end-of-course and 6-month follow-up evaluations show that participants enjoyed the training experience and believed it was valuable in helping them fulfill their leadership role as chief resident.

Adsorption with a soluble E. coli antigen fraction improves the specificity of ELISA tests for Lyme disease.

We reported that preadsorption of patient serum with heat killed E. coli increased the specificity of ELISA for antibodies to Borrelia burgdorferi. That procedure required extra specimen handling and a preincubation. We report the use of a soluble E. coli antigen fraction that is included in serum diluent, eliminating additional steps. Sera from 220 individuals were tested for antibodies to B. burgdorferi. Twenty sera were obtained from patients with Lyme disease and 200 sera were from a population that included healthy controls and patients with different inflammatory conditions (viral infections and various rheumatic disorders). Testing was performed using either a standard serum diluent or one containing soluble E. coli antigen fraction. Results demonstrate that inclusion of soluble E. coli antigen fraction in serum diluent increased assay specificity from 88% for the standard protocol to 98%, with no change in test sensitivity.

The pediatric program director. An analysis of the role and its problems.

A questionnaire designed to elicit information about the work environment, knowledge, and stresses of pediatric residency program directors was mailed to the 235 member programs of the Association of Pediatric Program Directors (APPD). At the time that the 187 respondents (80% return rate) assumed responsibility for their training programs, many rated their knowledge of various aspects of residency program administration as "poor." The respondents indicated that a lack of time, the pressures of too many other academic responsibilities, and a fear of not "filling" all positions in the National Intern Matching Program created much personal stress. Most program directors felt that educational conferences designed to teach educational methods, and administrative skills, and provide technical information knowledge necessary for residency program supervision would be beneficial.

Induction of humoral manifestations of autoimmunity following intraperitoneal injection of complete Freund's adjuvant in mice.

Several animal models of arthritis are produced using complete Freund's adjuvant (CFA) alone or with collagen as an arthritogen. Successful induction of arthritis is reported to require that the adjuvant mixture be administered by intradermal or subcutaneous routes. The resulting arthritis is caused by primarily cellular immune responses. Data presented in this paper show that giving CFA by intraperitoneal (I.P.) inoculation results in a humoral autoimmune response, with no obvious signs of arthritis. This humoral autoimmune response is characterized by production of autoantibodies to nuclear and cytoplasmic antigens, elevated levels of circulating immune complexes, and in approximately 25% of mice, rheumatoid factor.

An adsorption procedure to increase the specificity of enzyme-linked immunosorbent assays for Lyme disease without decreasing sensitivity.

Previous efforts to use adsorption techniques to enhance the specificity of enzyme-linked immunosorbent assays for antibodies to the spirochete that causes Lyme disease resulted in unacceptable reductions in assay sensitivity. We present here evidence that preadsorption of serum with Escherichia coli can enhance test specificity without significantly reducing test sensitivity.