Predicting duration of beneficial effect of joint injection among children with chronic arthritis by measuring biomarkers concentration in synovial fluid at the time of injection.

OBJECTIVES: Intra-articular corticosteroids injection (IAC) is a mainstay for the treatment of children with chronic arthritis; nonetheless its efficacy showed variability among published studies and it is still not possible to predict the outcome in a single patient. Our objective was to study the profile of biomarkers in the synovial fluid (SF) obtained at the time of injection and establish if such profile predicts duration of effect. METHODS: SF obtained from patients who underwent knee arthrocentesis and injection was procured and stored for cytokine analysis. Records of those patients who had at least 6 months of follow-up from the injection were reviewed. Time to flare was recorded. Levels of IL-6, IL-1alpha, TNF-alpha, IL-2sR, MMP-3, IL-10 and TGF-Beta1 were measured by ELISA. For primary analysis each patient was utilized once. For secondary analysis each injected knee was considered a single event. RESULTS: 60 samples from 33 patients were obtained. In the primary analysis we found a correlation between MMP-3 synovial fluid levels and outcome at 6 months (p=0,02; p=0,03 for different quartiles). In the secondary analysis we found that IL-6 and IL-10 levels predicted outcome at six and at 12 months (IL-6: p=0.01; p=0.02 respectively) (IL-10: p=0.017; p=0.01 respectively), with higher levels of IL-6 predicting shorter time to relapse and higher levels of IL-10 longer duration of corticosteroids effect. CONCLUSIONS: Our study identified MMP-3 and possibly IL-6 and IL-10 as candidates for the development of a set of biomarkers to predict response to IAC among children with chronic arthritis at the time of injection.

Arthritis following recombinant outer surface protein A vaccination for Lyme disease.

As more individuals receive outer surface protein A (OspA) vaccination, adverse effects not detected during phase III clinical trials may become apparent. Although arthritis has been described following other human vaccines, we found no reports of human cases after Lyme disease vaccination. We describe 4 males (2 children, 2 adults) who developed arthritis following recombinant OspA vaccination. The potential arthritogenic effect of OspA suggested by in vitro and animal studies finds a clinical correlate in these 4 cases.

Antibody response to IR6, a conserved immunodominant region of the VlsE lipoprotein, wanes rapidly after antibiotic treatment of Borrelia burgdorferi infection in experimental animals and in humans.

Invariable region (IR)(6), an immunodominant conserved region of VlsE, the antigenic variation protein of Borrelia burgdorferi, is currently used for the serologic diagnosis of Lyme disease in humans and canines. A longitudinal assessment of anti-IR(6) antibody levels in B. burgdorferi-infected rhesus monkeys revealed that this level diminished sharply after antibiotic treatment (within 25 weeks). In contrast, antibody levels to P39 and to whole-cell antigen extracts of B. burgdorferi either remained unchanged or diminished less. A longitudinal analysis in dogs yielded similar results. In humans, the anti-IR(6) antibody titer diminished by a factor of > or =4 in successfully treated patients and by a factor of <4 in treatment-resistant patients. This result suggests that the quantification of anti-IR(6) antibody titer as a function of time should be investigated further as a test to assess response to Lyme disease therapy or to determine whether a B. burgdorferi infection has been eliminated.

Effect of immunization with recombinant OspA on serologic tests for Lyme borreliosis.

This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to Borrelia burgdorferi. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with B. burgdorferi. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection with B. burgdorferi in OspA vaccine recipients.

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.

Helicobacter pylori can be induced to assume the morphology of Helicobacter heilmannii.

Cultures of Helicobacter pylori obtained from the American Type Culture Collection (strain 43504) were grown as isolated colonies or lawns on blood agar plates and in broth culture with constant shaking. Examination of bacterial growth with Gram-stained fixed preparation and differential interference contrast microscopy on wet preparations revealed that bacteria grown on blood agar plates had a morphology consistent with that normally reported for H. pylori whereas bacteria from broth cultures had the morphologic appearance of Helicobacter heilmannii. Bacteria harvested from blood agar plates assumed an H. heilmannii-like morphology when transferred to broth cultures, and bacteria from broth cultures grew with morphology typical of H. pylori when grown on blood agar plates. Analysis by PCR of bacteria isolated from blood agar plates and broth cultures indicated that a single strain of bacteria (H. pylori) was responsible for both morphologies.

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).

Pharmacokinetics of teratogenic antibodies administered to rats.

The serum levels of total and visceral yolk sac (VYS)-reactive sheep IgG have been determined following injection of a teratogenic sheep anti-VYS antiserum. After intravenous injection, levels of VYS-reactive IgG fell rapidly, with 75% of the amount in the injection removed in the first 5 min, and 90% by 60 min. By contrast, 90% or more of the total sheep IgG was still present at these times. A similar difference in clearance was seen after intraperitoneal injection, although the serum levels also reflected the presence of a pool of antiserum in the peritoneum and the simultaneous influx and efflux of IgG into and from the circulation. The clearance pattern was similar in pregnant and nonpregnant rats; it is concluded that antibodies against VYS-specific antigens comprise a very small fraction of VYS-reactive antibodies in the antiserum. ELISA and Western blot analysis indicated extensive cross-specificity with antigens present in rat tissues other than the VYS. Similar teratogenic effects were observed after intravenous or intraperitoneal injection of the antiserum at 8.5 days of gestation; we conclude that the proximal effect likely begins within the period immediately after the injection. The results are also considered within the context of published reports that the VYS shows structural and functional damage for several days after intraperitoneal administration of antiserum at 8.5 days.

Comparative evaluation of adsorption with E. coli on ELISA tests for Lyme borreliosis.

OBJECTIVE: To evaluate prospectively in a clinical setting the use of a soluble fraction of E. coli to adsorb nonspecific antibodies which can cause false positive ELISA tests for Lyme borreliosis. METHODS: The patient population tested was obtained from individuals referred to or initially presenting at a pediatric Lyme disease clinic in Wilmington, DE. Patients were followed for a minimum of 6 months subsequent to primary presentation at the clinic. RESULTS: A total of 209 met criteria for study inclusion, 93 of whom were diagnosed as having Lyme borreliosis and 116 of whom had other diagnoses. Results of ELISA tests were compared with different diagnoses and, when available, ELISA results from commercial laboratories. Findings indicate that some commercial laboratories have excessively high rates of false positive results (> 90% of positives were found to be false positives). CONCLUSION: Adsorption with E. coli antigens effectively removed antibodies causing false positive results including those occurring at commercial laboratories and did not cause any significant reduction in assay sensitivity.

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.

Examination of anti-neutrophil cytoplasmic antibodies in childhood inflammatory bowel disease.

The detection of anti-neutrophil cytoplasmic antibodies (ANCA), in a perinuclear fluorescence pattern, in the serum of adults with inflammatory bowel disease has recently been described to be sensitive and specific for a diagnosis of ulcerative colitis in comparison to Crohn's disease and other colitides. We have examined the sera of 41 children and adolescents with ulcerative colitis, 27 with Crohn's disease, and a control group for the presence of ANCA. Anti-neutrophil cytoplasmic antibodies were detected in the serum of 27 of 41 patients with ulcerative colitis (66%), five of 27 with Crohn's disease (19%), and in none of our control subjects or patients with functional abdominal pain. Overall, the presence of ANCA was 66% sensitive and 84% specific for a diagnosis of ulcerative colitis when compared to Crohn's disease. There was no relationship between a positive ANCA value and disease activity or other clinical indicators. We conclude that evaluation for the presence of ANCA may be a useful adjunct for the clinical assessment of patients with inflammatory bowel disease. The presence of ANCA in children and adolescents, however, will not definitively distinguish between patients with ulcerative colitis and Crohn's disease.

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.

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)