Diagnosis of lyme borreliosis in europe

In Europe, Lyme borreliosis is caused by at least three species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. Thus microbiological diagnosis in European patients must consider the heterogeneity of Lyme disease borreliae for development of diagnostic tools such as PCR primers and diagnostic antigens. According to guidelines of the German Society of Hygiene and Microbiology, the serological diagnosis should follow the principle of a two-step procedure. A sensitive ELISA differentiating IgM and IgG is recommended as the first step. In case the ELISA is reactive, it is followed by immunoblots (IgM and IgG) as the second step. The reactive diagnostic bands should be clearly identified, which is easy if recombinant antigens are used. The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens instead of whole cell lysates. Improved sensitivity resulted from use of recombinant proteins that are expressed primarily in vivo (e.g., VlsE) and combination of homologous proteins from different strains of borrelia (e.g., DbpA). It also appears promising to use recombinant proteins (DbpA, VlsE, others) or synthetic peptides (the conserved C6 peptide derived from VlsE) as ELISA antigens. At present, detection rates for serum antibodies are 20-50% in stage I, 70-90% in stage II, and nearly 100% in stage III Lyme disease. The main goals for the future are to improve specificity in general and sensitivity for diagnosis of early manifestations (stage I and II). Detection of the etiological agent by culture or PCR should be confined to specific indications and specialised laboratories. Recommended specimens are skin biopsy specimens, CSF and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50-70%) and synovial tissue or fluid (50-70% with PCR). CSF yields positive results in only 10-30% of patients. Methods that are not recommended for diagnostic purposes are antigen tests in body fluids, PCR of urine, and lymphocyte transformation tests.     

Serodiagnosis of Rocky Mountain spotted fever: comparison of IgM and IgG enzyme-linked immunosorbent assays and indirect fluorescent antibody test

To characterize IgM and IgG antibody responses in Rocky Mountain spotted fever (RMSF), a microtiter enzyme-linked immunosorbent assay (ELISA) using density gradient-purified Rickettsia rickettsii as antigen was developed. Sera of vaccinated individuals and patients with RMSF were tested by ELISA and by indirect fluorescent antibody (IFA) tests. Diagnostic agreement between ELISA and the IFA test was 76% and 52% for IgG and IgM antibody, respectively. Diagnostic agreement between the ELISA for IgG antibody and the IFA test for total immunoglobulins was 84%. The ELISAs for IgM and IgG antibody were as specific (100%) and as sensitive (100%) as the IFA test (83%-100%) in detecting antibody increases in paired sera from persons with RMSF and were superior to the IFA test in detecting seroconversions in vaccinees. The ELISA also detected antibodies in a single convalescent-phase serum with sensitivity and reliability. The ELISA for IgG antibody is appropriate for seroepidemiology and serodiagnosis since it permits measurement of antibody at a single dilution of serum up to a year after illness.     

Comparative evaluation of selected diagnostic assays for the detection of IgG and IgM antibody to Orientia tsutsugamushi in Thailand

We compared the performance of 2 commercially available dipstick assays, 2 enzyme-linked immunosorbent assays (ELISAs), and an indirect immunofluorescent antibody (IFA) assay for the diagnosis of scrub typhus, using the indirect immunoperoxidase (IIP) test as the reference standard. The dipstick assays were the Integrated Diagnostics (Baltimore, MD) Dip-S-Ticks Scrub Recombinant (r56) dipstick test (INDX assay) and the PanBio (Brisbane, Australia) Scrub Typhus IgM and IgG Rapid Immunochromatographic test (PanBio assay). One of the ELISAs used pooled cell lysates of Karp, Kato, and Gilliam strain Orientia tsutsugamushi as antigen (pooled-antigen ELISA), and the other used a recombinant r56 protein as the antigen (recombinant ELISA). With a panel of 123 positive and 227 negative sera, sensitivity and specificity of the assays were as follows: INDX assay, IgG, 60% and 95%, IgM, 60% and 97%; PanBio assay, IgG, 94% and 96%, IgM, 83% and 93%; IFA (1:400 cutoff), IgG, 91% and 96%, IgM, 85% and 98%; pooled-antigen ELISA, IgG (1:1600 cutoff), 97% and 89%, IgM (1:400 cutoff), 94% and 91%; recombinant ELISA, IgG (1:1600 cutoff), 97% and 92%, IgM (1:400 cutoff), 93% and 94%. Because of its excellent performance and use of a standardized, commercially available antigen, the recombinant ELISA is suitable for use in a diagnostic laboratory, where it may be able to replace the IFA and IIP assays. In contrast, the PanBio dipstick assay was easy to perform and did not require sophisticated equipment, making it suitable for use in rural areas where more sophisticated diagnostic tests such as the ELISA and IFA may not be available.     

Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates

In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent.     

中国B.afzelii基因型莱姆病螺旋体GDsh1表面蛋白VlsE保守区段的克隆表达及抗原性分析

目的 克隆表达中国莱姆病螺旋体B.afzelii基因型菌株GDsh1的表面蛋白VlsE保守区段,并对其抗原性进行分析,为制备中国莱姆病重组抗原ELISA检测试剂盒提供依据。方法 结合文献,下载并比对PubMed上所有莱姆病螺旋体B.a型菌株的VlsE基因序列,确定保守区段,设计引物,扩增GDsh1 的VlsE基因片段。将扩增产物与载体PET-32a连接,转入大肠杆菌BL21(DE3)中表达。对重组载体进行序列测定,表达产物用SDS-PAGE和Western blot 分析。利用重组VlsE蛋白制备ELISA试剂盒,检测83份莱姆病阳性血清,90份阴性血清以及90份梅毒血清,计算重组试剂盒的灵敏度和特异性。并与科室已有的全菌蛋白ELISA试剂盒检测结果进行比较。结果 成功克隆表达了B.afzelii型VlsE保守区蛋白,Western blot结果显示VlsE保守区蛋白与免疫兔血清有较强的抗原抗体反应。ELISA结果表明:重组VlsE蛋白的灵敏度60.2%低于全菌蛋白的灵敏度92.8%(P<0.001);特异性分别为73.3%、68.9%,差异无统计学意义(P=0.511)。在检测梅毒血清上特异性分别为83.3%、18.9%,重组蛋白的特异性远远高于全菌蛋白(P<0.001)。结论 重组VlsE基因保守区段蛋白在莱姆病的检测中具有一定的灵敏度和特异性,且在区分梅毒血清与莱姆病血清上,其特异性远远高于全菌蛋白,在莱姆病血清学检测中具有不容忽视的重要意义。     

酶联免疫吸附试验检测人血清弓形体IgG,IgM抗体的研究

对ELISA检测人血清弓形体IgG、IgM抗体进行了研究。450份孕妇血清中,ELISA阳性率显著高于IHA;抗体滴度分折,ELISA一般高于IHA2～10倍。25份ELISA IgG抗体阳性血清,IFA检出19份;3份IgM抗体阳性和2份IgG、IgM抗体均阳性血清,IFA分别检出2份。2份阴性和4份含不同抗体滴度的阳性血清于第一次测定后,第7天和第21天测定的阴、阳性结果一致,OD值变异系数为2.43～16.52％。39份阳性血清抗体滴度与OD值呈直线比例关系(r=0.991,P<0.0005)。结果表明,ELISA用于弓形体感染的血清学诊断具有较好的实用性。     

Persistence of serum antibodies to Borrelia burgdorferi in patients treated for Lyme disease.

To determine if antibodies to Borrelia burgdorferi persist after antibiotic treatment, we recalled 32 patients with Lyme disease from a primary care practice a mean of 16 months after treatment and analyzed initial and follow-up serum samples by ELISA and immunoblot assays. Of the eight patients whose initial serum specimens were positive for IgM antibody by ELISA, three had positive titers of IgM antibody at follow-up; of the 23 patients whose initial serum specimens were positive for IgG antibody by ELISA, 19 had positive titers of IgG at follow-up. Of the five patients whose initial serum specimens were positive for IgM antibody by immunoblot, two had positive titers of IgM antibody at follow-up; of the 30 patients whose initial serum specimens were positive for IgG antibody by immunoblot, 29 had positive titers of IgG antibody at follow-up. The bands on the IgG immunoblot remained remarkably constant during the period from analysis of the initial specimen to that of the follow-up specimen. Nine of the 32 patients had persistent or recurrent symptoms, and ELISA and immunoblot were not helpful for identifying these nine patients.     

Evaluation of enzyme-linked immunosorbent assay for Chlamydophila pneumoniae-specific immunoglobulin M in acute respiratory tract infection

Background and objective:   The study evaluated a newly developed ELISA (Hitazyme Chlamydophila pneumoniae ) for detecting anti- C. pneumoniae -specific IgM antibody, by comparing the ELISA assay to a microimmunofluorescence (MIF) test and immunoblotting.
Methods:   One hundred patients with acute respiratory tract infections (58 children and 42 adults) were enrolled in the study. Paired sera were obtained from all subjects for serological testing of C. pneumoniae .
Results:   C. pneumoniae IgM positivity was observed in 36 (62.0%) children and 11 (26.1%) adults. However, MIF test or immunoblot revealed only four positive reactions in these patients. These four IgM-positive patients were also positive by ELISA. A significant increase in IgG and/or IgA antibody titres in paired sera was observed in three of the four patients. Of the remaining 96 patients, no significant increase in IgG or IgA antibody titre in the paired sera was observed. To confirm the positive reactivity of ELISA, positive sera were also analysed by recombinant enzyme immunoassay. Forty-three cases that were IgM-positive only by ELISA were all negative by recombinant enzyme immunoassay and the ELISA results were considered to be false-positives.
Conclusions:   These results indicate that a newly developed ELISA for detecting anti- C. pneumoniae -specific IgM antibody frequently generates false-positive findings in patients with acute respiratory tract infections, at the current cut-off level. Further studies are needed to determine an appropriate cut-off level and the possible causes of the false-positive results in the ELISA.     

Recombinant or peptide antigens in the serology of Lyme arthritis in children

The performance of ELISAs with the recombinant antigens decorin-binding protein A (DbpA), DbpB, and BBK32 (from Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto) and VlsE peptide antigen invariable region 6 (IR(6)) were evaluated in the serodiagnosis and follow-up of children with Lyme arthritis (LA). Serum samples were obtained from 52 children with clinically typical and serologically confirmed LA. In IgG ELISAs, at diagnosis, 50 samples were positive for BBK32, 51 for DbpA, 40 for DbpB, and 51 for IR(6). In the posttreatment follow-up, the rate of decline of the antibodies to the recombinant protein antigens or to IR(6) did not appear useful in the prediction of the treatment response or the clinical course of LA. Yet, IR(6) seems to have the greatest potential to be used universally in the diagnostic serology of Lyme borreliosis (LB). Alternate to that, the use of several specific borrelial antigens, in parallel, might improve the accuracy of serology for LB.     

Clinical evaluation of commercial serological test for Bartonella infection

We evaluated the usefulness of a serological diagnostic kit (Bartonella IFA IgG, IgM; MRL Diagnostics) for Bartonella henselae infection. Of the 110 healthy individuals, 107 (97.3%) were with titers being less than 1:64 for IgG antibody to B. henselae, 2 were with titers being 1:64 and 1 with 1:128, IgM antibody to B. henselae was negative in all individuals. Serological diagnosis of cat scratch disease (CSD) using indirect fluorescence antibody (IFA) methods (in-house and diagnostic kit) was made in either elevated titers of IgM (> or = 1:20) or IgG (> or = 1:256) antibodies, or a four-fold rise in IgG titer between acute and convalescent sera. Of the 18 individuals with serological diagnosis of CSD by in-house IFA method in 26 CSD clinical diagnosed patients, 15 (83%) were compatible with the results of the diagnostic kit, whereas 3 (17%) were not compatible. Of the 8 without serological diagnosis, 1 (13%) was serologically diagnosed as CSD, and the others were negative. Overall, the serological diagnosis was made in 16 of 26 (62%). The specificity and sensitivity of this kit were 100% and 62%, respectively. The cross-reaction between B. henselae and Bartonella quintana was observed in sera from controls and patients. Our results show that the diagnostic kit as well as in-house method is an useful tool for the serological diagnosis of cat scratch disease.     

Antibody response to human immunodeficiency virus after primary infection

The antibody response to human immunodeficiency virus (HIV) after primary infection was monitored in eight homosexual men with the acute mononucleosis-like illness associated with seroconversion. Multiple sera from each subject, taken at frequent intervals after onset of acute illness, were tested for antibody to HIV by IgM and IgG immunofluorescent assays (IFAs), four commercial enzyme-linked immunosorbent assays (ELISAs), and Western immunoblot (WB). Antibody to HIV was detected first by IgM IFA (mean +/- SD, 5 +/- 3 days), followed by IgG IFA (11 +/- 3 days); the IgM antibody titer peaked at 24 +/- 17 days and disappeared by 81 +/- 27 days, whereas the IgG antibody titer peaked at 133 +/- 63 days and has not disappeared in any subject. Antibody to HIV was first detected by ELISA from 31 +/- 14 to 58 +/- 32 days, depending on the assay kit used. Antibody to p24 and gp41 was first detected by WB at 24 +/- 10 days, followed by antibody to p55 (40 +/- 20 days), p68 (57 +/- 19 days), and p34 (71 +/- 22 days).     

IgM rheumatoid factor in Lyme disease: correlation with disease activity, total serum IgM, and IgM antibody to Borrelia burgdorferi

We tested the sera of 50 patients with Lyme disease for IgM-rheumatoid factor (IgM-RF) using a sensitive ELISA. Levels of IgM-RF greater than 3 SD above the mean of normal subjects were found in 2 of 15 patients with erythema chronicum migrans, 7 of 10 with neurologic abnormalities, and 7 of 25 with Lyme arthritis (p = 0.038). Only 2 of these sera were positive by latex agglutination. In contrast, none of the 23 control patients with osteoarthritis, ankylosing spondylitis, or Reiter's syndrome had positive tests. The levels of IgM-RF correlated with disease activity (p = 0.002), total serum IgM levels (p = 0.002), and specific IgM antibody titers to Borrelia burgdorferi (p = 0.006). IgM-RF reactivity was absorbed with heat aggregated IgG (HAGG), but the titer of specific IgM antibody was insignificantly affected by this procedure. Thus, small amounts of RF are produced at certain times in many patients with Lyme disease, and IgM-RF production appears to be linked to the specific IgM response.     

Antibody response in Lyme disease: evaluation of diagnostic tests

The antibody response to the Ixodes dammini spirochete was determined in 41 serial serum samples from 12 patients with Lyme disease. By enzyme-linked immunosorbent assay (ELISA), 11 of the 12 patients had higher titers of specific IgM antibody (greater than 1:200) during early disease than did 40 control subjects. Specific IgM antibody titers, which correlated with total amounts of IgM antibody (P less than .001), sometimes remained elevated throughout the illness. During neuritis, nine of 10 patients had higher specific IgG antibody titers (greater than 1:200) than did controls, and when arthritis was present, all had such titers, which remained elevated after months of remission. In the ELISA, antibody responses determined by single or serial dilutions were similar, but the ELISA was more sensitive and specific than was immunofluorescence. Adsorption of sera with Borrelia hermsii generally resulted in a fourfold decrease in titers of cross-reactive antibodies, but the titers of sera from patients with Lyme disease were also reduced. Currently, the ELISA, without adsorption, is the best diagnostic test for Lyme disease.     

Differential antibody responses to rubella virus infection in males and females.

Specificities of human rubella virus (RV)-specific IgG, IgM, and IgA antibodies for RV structural proteins (envelope E1 and E2 and capsid) and an E1 domain represented by a synthetic peptide (BCH-178) were determined by immunoblot and ELISA techniques in sera from rubella-infected individuals. Sequential sera were obtained from 67 females and 32 males during acute and convalescent infection phases. Males and females had differences in all antibody classes, especially during the early acute infection phase. At no time during follow-up were IgA anti-E2 antibodies detected in males, in contrast to the case in females. Significantly lower levels of IgG antibody directed to E2 were also observed in males. Males had earlier onset of E1-specific IgG and IgM antibodies with a greater proportion of total RV antibody response directed to E1 (IgG) or E1 peptide (IgM). These differences suggest there are hormonal and genetic influences on immune recognition of RV proteins that may be related to the increased incidence of rubella-associated arthropathy in females.     

用不同基因型戊型肝炎病毒重组蛋白建立酶联免疫吸附测定一步法

目的 以7种不同基因型和亚型的HEV重组蛋白作为包被抗原,建立HEV抗体检测ELISA一步法.方法 通过方阵滴定法确定包被抗原浓度,确定临界值,并进行敏感度、特异度和热稳定性试验.结果 7种重组抗原混合物(Mix166)最佳包被浓度为1.5 mg/L.抗-HEV IgG检测试剂的批内、批间变异系数分别为8.67%和10.85%,抗-HEV IgM检测试剂的批内、批间变异系数分别为4.56%和5.99%.一步法检测50份HEV RNA阳性血清的HEV IgG和HEV IgM抗体,阳性率均为94%.一步法检测674份健康者血清,52份抗-HEV IgG阳性,3份抗-HEV IgM阳性.一步法检测HEV墨西哥株攻击黑猩猩后收集的系列血清发现,病毒攻击后1～6周抗-HEVIgM阳性,2～76周抗-HEV IgG阳性,而进口试剂盒缺乏对抗-HEV墨西哥株IgG、IgM的反应性.结论 Mix166作为包被抗原建立的HEV抗体ELISA一步法具有较好的敏感性和特异性,可用于HEV感染的诊断.     

Prevalence of antibody to Borrelia burgdorferi by indirect fluorescent antibody assay, ELISA, and western immunoblot in healthy adults in Wisconsin and Arizona.

The prevalence of antibody to Borrelia burgdorferi in healthy adults from Wisconsin and Arizona was determined by indirect fluorescent antibody assay (IFA), ELISA, and Western immunoblotting. A total of 301 sera from adult volunteer blood donors were collected from three areas of Wisconsin and compared with 49 consecutive anonymous adult volunteer donor sera from Tucson, Arizona, an area without reported Lyme borreliosis. Regional differences in seropositivity were found for Western immunoblotting (34[11%] of 301 from Wisconsin and none of 49 from Tucson; P less than .01) but not IFA or ELISA. No correlation was found among Western immunoblotting and IFA or ELISA results. For persons living in Madison or Milwaukee, Wisconsin (cities not endemic for Lyme borreliosis), 19 (86%) of 22 with a positive Western blot, but only 12 (48%) of 25 with a positive IFA or ELISA, had a significant exposure risk to B. burgdorferi-infected Ixodes dammini (odds ratio, 6.9; 95% confidence interval, 1.4-43.5). Western blot results were consistent with epidemiologic exposure to B. burgdorferi and implied frequent asymptomatic infection among healthy adults living in or visiting areas endemic for Lyme borreliosis.     

A limitation of 2-stage serological testing for Lyme disease: enzyme immunoassay and immunoblot assay are not independent tests.

To improve the accuracy of testing for antibody to Borrelia burgdorferi, 2-stage conditional testing has been recommended, in which sera that yield positive or equivocal results in a first-stage test (e.g., an ELISA) are then tested by immunoblot assay. The increased specificity anticipated with sequential testing, however, depends on immunoblot assays and ELISAs being independent tests. To examine whether they are independent, control serum samples were tested with 2 different commercially available IgM ELISAs and with an IgM immunoblot assay kit. The frequency of false-positive IgM immunoblot assays was significantly higher with ELISA-reactive than with ELISA-negative serum samples (P相似文献   

Enzyme-linked immunosorbent assay and indirect immunofluorescence assay for Lyme disease

The sensitivity and specificity of an indirect immunofluorescence assay (IFA) and ELISA for Lyme disease were estimated. Sera from patients with Lyme disease, patients with other infections, and healthy individuals were examined. Significant cross-reactivity occurred only with sera from patients with syphilis, yaws, and pinta . All tested sera from patients with Lyme disease, however, gave negative results in the rapid reagin screening test and the microhemagglutination assay for antibodies to Treponema pallidum confirmatory for syphilis. When sera from patients with treponemal diseases were excluded from the analysis, the IFA and ELISA were highly specific, having 97% and 100% reliability, respectively. Sensitivity of both tests varied with the stage of disease but was 100% for both tests during complicated Lyme disease. The results indicate that both tests are highly specific and sensitive for complicated Lyme disease but relatively insensitive for patients with erythema chronicum migrans alone.     

Assessment of assays for the serodiagnosis of Venezuelan equine encephalitis.

An enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA), a plaque-reduction neutralization (PRN) assay and an immunoblot assay, all by means of an antigen prepared from the attenuated Venezuelan equine encephalitis (VEE) vaccine strain of virus, were compared with the conventional haemagglutination-inhibition (HAI) assay for the serodiagnosis of VEE. The HAI assay, which includes the use of wild type virus antigen, was less sensitive than the other assays when known-positive samples of serum from an epidemic of VEE were tested. The superior sensitivity of the IgG ELISA was confirmed by assaying both VEE epidemic samples and a bank of samples from VEE vaccinees. Samples with antibody specific for other Alphaviruses, however, cross reacted weakly in this assay. The PRN, immunoblot and HAI assays, although less sensitive than the ELISA, proved more specific. Experimental infection of guinea-pigs demonstrated the value of the IgM ELISA in the early detection of VEE virus infection. Immunoglobulin M was first found at 4 days post-inoculation (p.i.) during the viraemic phase of infection. Immunoglobulin G was detected by ELISA, PRN assay and IFA at 6 days p.i. Immunoblot and HAI assays, however, did not give positive results until 10 days p.i. The results support the diagnostic use of ELISA for detecting VEE virus-specific IgM and IgG, and the use of the specific PRN assay for confirming the diagnosis.     

棘球蚴病患者IgG抗体阴性反应血清再检测的研究

目的　探索棘球蚴病患者抗体应答假阴性反应原因 ,以改进棘球蚴病的免疫诊断方法。　方法　采用间接ELISA和双抗体夹心ELISA方法 ,检测 42例IgG抗体阴性反应棘球蚴病患者血清的IgG亚类 (IgG1、IgG2、IgG3和IgG4 )、IgA、IgM、IgE抗体及抗原和循环免疫复合物。　 结果　 42例阴性血清中 ,32例IgG亚类或IgA、IgM、IgE抗体阳性 ,1 0例血清抗体全部阴性。其中IgG1、IgG4及IgA、IgM、IgE的检出率明显高于正常人 ,分别为 42 .9%、1 1 .9%、2 8.6 %、2 6 .2 %和 2 1 .4 %。小儿的IgM高于成人。肝棘球蚴病患者的IgG亚类高于肺棘球蚴病患者。IgG1与其它抗体联合检测 ,以IgG1 +IgA +IgM检出率最高 ,为 64 .3 %。IgG阴性患者血清的CAg和CIC阳性率分别为 2 8.57%及30 .95 %。　结论　抗棘球蚴总IgG抗体表达水平低下 ,抗体表达种类不同及循环免疫复合物的形成 ,是造成棘球蚴病患者IgG抗体反应阴性的主要原因。IgG1 +IgA +IgM检测可提高棘球蚴病患者的诊断率