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How Accurate Is The Lyme Disease Elisa Test?

The most common position mentioned in books and journals is the use of the ELISA test to "screen" to see if someone has Lyme disease. No mention is made of other infections, so it appears the belief is that you must have Lyme disease to have other infections carried by insects.

Of course this is wrong, since Bartonella can be carried by vastly more infectious insects, and Bartonella can suppress Lyme antibodies. But this is not really appreciated by those with a "Lyme alone" focus.

First, it is clear the ELISA type of test can be very useful. For example, Dr. Hooper has developed a very high quality test for indoor and warfare mold biotoxins that has never been ejected from a court. Currently, we have 33,000 articles published on mycotoxins so this is hardly new or tangential medicine—it is just not a medical ill requiring a pharmaceutical product so physicians never hear about it.

It seems some feel the ELISA is an exceptional test for screening for Lyme disease. Others feel it is poor. Others do not give it a thought.

The issue of what lab runs the test occasionally is mentioned but not much, because it is well known if you send tubes to misc labs, you get different results.

My only appeal, is one based on gambling. I do not think you gamble in medicine, serious money or with your liberty. For example, we have two very elderly pro jail judges, who demand speed trials in front of a mere six people—I mean murder, rape and arson cases are in front of six people. Government leaders and lawyers report these jurors are intellectually "limited." I saw this when I served in the jury pool. So here a trial is like playing with TNT.

Lyme disease and the other infections that are usually present, can present with zero symptoms, which is the rule with 95% of dogs (Susta and Uhl). And my concern in humans is that when a person has real symptoms, it is often not their first bite and using one test, when tick infections impact 15 plus indirectly is perhaps a bit too much faith. My appeal is we should not have 100% faith in any aspect of science except basic and proven math, not emerging infection lab tests.

But ultimately you have to decide if the ELISA test for the most discussed human species is enough. Or do you want wider testing, because if you miss a case, a child or patient may die. Further, treating for years with antibiotics also seems unusual and something done by some almost impulsively. Surely, in 2012, we can do better.

Below are many articles on the ELISA and this infection. Glance over it knowing that most clinical experience never makes it to a publication. I know I only publish 3% of what we know and do.

My thanks to all those who sacrificed to share this information below. It is no fun and never easy.

1. Vet Pathol. 2011 Nov 10. [Epub ahead of print]

Synovial Lesions in Experimental Canine Lyme Borreliosis.

Susta L, Uhl EW, Grosenbaugh DA, Krimer PM.

Borrelia burgdorferi is the causative agent of Lyme disease, which is mainly characterized by lameness in dogs. More than 95% of naturally infected dogs are asymptomatic or subclinical; however, in experimental studies, histologic synovial lesions are consistently observed in asymptomatic dogs inoculated with B. burdgorferi. This study investigates the ability of a synovial histopathologic scoring system, clinicopathologic data, and polymerase chain reaction (PCR) testing to differentiate between B. burgdorferi-infected and uninfected dogs. Eighteen 18-week-old beagles were subject to challenge with B. burgdorferi-infected wild-caught ticks (Ixodes scapularis), and 4 uninfected dogs served as controls. Infection was confirmed by serology (ELISA) and PCR amplification of B. burgdorferi-specific DNA of skin biopsies taken at the tick attachment site. A synovial scoring system from human medicine was adapted and implemented on postmortem synovial samples to discriminate infected and noninfected animals. Application of this system to elbows and stifles with a cumulative joint score cutoff > 4 showed a sensitivity of 88.2% and a specificity of 100%, with a positive likelihood ratio of infinity and a negative likelihood ratio of 0.12. Complete blood count, serum biochemistry, urinalysis, urine protein:creatinine, urine PCR, synovial and lymph node cytology, and synovial PCR were evaluated but were not reliable indicators of clinical disease.

PMID: 22075774 [PubMed - as supplied by publisher]

2. J Fr Ophtalmol. 2011 May;34(5):325.e1-3. Epub 2011 Apr 14.

[Acquired nystagmus in a 12-year-old boy as initial presentation of Lyme disease].

[Article in French]

Samimi S, Salah S, Bonicel P.

Service d'ophtalmologie, centre hospitalier régional d'Orléans, 14, avenue de l'Hôpital, 45067 Orléans cedex 02, France.

We report the case of a 12-year-old boy presenting with acquired horizontal nystagmus, headaches, and vertigo. CT, MRI, viral tests, and the Lyme disease test were at first negative. We made the diagnosis of neuroborreliosis based on a repeated Lyme disease test and lumbar puncture revealing intrathecal synthesis of specific antibodies. Adjusted antibiotic treatment led to complete disappearance of symptoms. Lyme borreliosis is difficult to diagnose and should be sought in case of unusual neuro-ophthalmic signs, especially in children.

Copyright © 2011 Elsevier Masson SAS. All rights reserved.

PMID: 21496946 [PubMed - indexed for MEDLINE]

3. Neurology. 2011 Mar 22;76(12):1051-8.

A prospective study on the role of CXCL13 in Lyme neuroborreliosis.

Schmidt C, Plate A, Angele B, Pfister HW, Wick M, Koedel U, Rupprecht TA.

Department of Neurology, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany.

Comment in Nat Rev Neurol. 2011 May;7(5):244. Neurology. 2011 Mar 22;76(12):1034-5.

BACKGROUND: The definite diagnosis of acute Lyme neuroborreliosis (LNB) requires detection of an increased Borrelia burgdorferi-specific antibody index (AI). The B burgdorferi AI, however, is negative in up to 20% of patients with early LNB and can remain elevated for years after adequate therapy; both of these factors can make the diagnosis difficult. Recent retrospective studies suggested the chemokine CXCL13 as a potential biomarker for LNB. To evaluate its diagnostic value, we conducted a prospective study.

METHODS: From March 2008 to August 2009, CSF and serum samples from all patients in whom a B burgdorferi-specific AI was requested (n=692) and CSF analysis revealed CSF pleocytosis (n=192) were included in the study. Because of the low number of patients with untreated LNB, 13 additional retrospectively selected samples of patients with untreated LNB were added. CXCL13 concentrations were measured by ELISA and receiver operating characteristic curves were generated.

RESULTS: CSF CXCL13 was highly elevated in all patients with untreated acute LNB (mean=15,149 pg/mL) compared with that in the patients without LNB (mean=247 pg/mL). At a cutoff of 1,229 pg/mL, the sensitivity of CXCL13 was 94.1%, which is higher than the AI (85.7%). Only 7 patients (5 with a CNS lymphoma and 2 with bacterial meningitis) had a CXCL13 level above the cutoff, resulting in a specificity equal to the AI of 96.1%.

CONCLUSIONS: CXCL13 shows high sensitivity and specificity for acute, untreated LNB. This novel marker appears to be helpful in clinically atypical cases and, in particular, in early stages of the disease when the B burgdorferi AI is (still) negative.

© 2011 by AAN Enterprises, Inc.

PMID: 21422457 [PubMed - indexed for MEDLINE]

4. Eur J Clin Microbiol Infect Dis. 2011 Aug;30(8):1027-32. Epub 2011 Jan 27.

Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots.

Ang CW, Notermans DW, Hommes M, Simoons-Smit AM, Herremans T.

VUMC, Amsterdam, The Netherlands.

Comment in Eur J Clin Microbiol Infect Dis. 2011 Aug;30(8):1033-4; author reply 1035-7.

We investigated the influence of assay choice on the results in a two-tier testing algorithm for the detection of anti-Borrelia antibodies. Eighty-nine serum samples from clinically well-defined patients were tested in eight different enzyme-linked immunosorbent assay (ELISA) systems based on whole-cell antigens, whole-cell antigens supplemented with VlsE and assays using exclusively recombinant proteins. A subset of samples was tested in five immunoblots: one whole-cell blot, one whole-cell blot supplemented with VlsE and three recombinant blots. The number of IgM- and/or IgG-positive ELISA results in the group of patients suspected of Borrelia infection ranged from 34 to 59%. The percentage of positives in cross-reactivity controls ranged from 0 to 38%. Comparison of immunoblots yielded large differences in inter-test agreement and showed, at best, a moderate agreement between tests. Remarkably, some immunoblots gave positive results in samples that had been tested negative by all eight ELISAs. The percentage of positive blots following a positive ELISA result depended heavily on the choice of ELISA-immunoblot combination. We conclude that the assays used to detect anti-Borrelia antibodies have widely divergent sensitivity and specificity. The choice of ELISA-immunoblot combination severely influences the number of positive results, making the exchange of test results between laboratories with different methodologies hazardous.

PMCID: PMC3132383 PMID: 21271270 [PubMed - indexed for MEDLINE]

5. Arch Immunol Ther Exp (Warsz). 2011 Feb;59(1):69-77. Epub 2011 Jan 22.

Serodiagnosis of borreliosis: indirect immunofluorescence assay, enzyme-linked immunosorbent assay and immunoblotting.

Wojciechowska-Koszko I, Mączyńska I, Szych Z, Giedrys-Kalemba S.

Department of Microbiology and Immunology, Pomeranian Medical University, Powstańców Wielkopolskich 72, 70-111, Szczecin, Poland.

Lyme disease is an infectious, multi-system, tick-borne disease caused by genospecies of Borrelia burgdorferi bacteria sensu lato, characterized by remarkable heterogeneity. In this situation choosing an optimal antigen array for diagnostic tests seems problematic. The serological tests for borrelia routinely done in laboratories often produce ambiguous results, which makes a proper diagnosis rather complicated and thus delays the implementation of an appropriate treatment regimen. Thirty-seven outpatients and eight inpatients with suspected borreliosis diagnosis hospitalized at the Clinics of the Pomeranian Medical University (Szczecin, Poland), participated in the study. In order to detect the antibodies against Borrelia sensu lato three kinds of serological tests were used: indirect immunofluorescence assay (IIFA), enzyme-linked immunosorbent assay (ELISA), and immunoblot. The IIFA and immunoblot tests conducted on 45 patients (100%) produced positive results for both the IgM and IgG antibody types. In the case of ELISA, positive or borderline results were observed in only 24 patients (53.3%). The immunoblot test for IgM most frequently detected antibodies against the outer surface protein C (OspC) antigen (p25), and, in the case of IgG, against the recombinant variable surface antigen (VlsE). The IIFA screening test used for diagnosing Lyme borreliosis produced the highest percentage of positive results, which were then confirmed by immunoblot, but not by ELISA. Therefore using only ELISA as a screening test or for diagnosing Lyme borreliosis seems debatable.

PMID: 21258869 [PubMed - indexed for MEDLINE]

6. Clin Vaccine Immunol. 2011 Mar;18(3):406-13. Epub 2010 Dec 22.

BBK07 immunodominant peptides as serodiagnostic markers of Lyme disease.

Coleman AS, Rossmann E, Yang X, Song H, Lamichhane CM, Iyer R, Schwartz I, Pal U.

Department of Veterinary Medicine, Building 795, Room 1341, University of Maryland, College Park, MD 20742, USA.

Lyme disease (LD) is a tick-borne infection caused by the bacterial pathogen Borrelia burgdorferi. Current diagnostic tests mostly use borrelial lysates or select antigens to detect serum antibodies against B. burgdorferi. These immunoassays are not entirely effective, especially for detection of early infection. We have recently characterized an in vivo-induced antigen, BBK07, as a serodiagnostic marker for LD. We now report that in a line blot assay, recombinant BBK07 protein-based detection is 90% sensitive and nearly 100% specific against B. burgdorferi infection in humans. Using an overlapping peptide library of 23 peptides encompassing full-length BBK07, we identified the immunodominant epitopes of BBK07 during human infection. We show that a select combination of amino-terminal peptides significantly enhanced BBK07-based diagnostic accuracy compared to that with the full-length protein. Although in enzyme-linked immunosorbent assay (ELISA) studies BBK07 peptides had overall lower sensitivity than established serodiagnostic peptides, such as the VlsE peptide C6 and OspC peptide pepC10, for the detection of early human LD, a subset of serum samples that failed to recognize either VlsE or OspC peptides were preferentially reactive to BBK07 peptides. These results highlight the fact that BBK07 peptides could be useful to complement the efficacy of VlsE and OspC peptide-based serodiagnostic assays. Finally, using a panel of canine sera, we show that BBK07 peptide is also effective for LD diagnosis in infected dogs. Together, our data show that peptides from the B. burgdorferi surface protein BBK07 are highly specific and sensitive serodiagnostic markers, and we suggest their future use in LD diagnostic assays.

PMCID: PMC3067378 PMID: 21177911 [PubMed - indexed for MEDLINE]

7. BMC Infect Dis. 2010 Nov 1;10:317.

Utilization of serology for the diagnosis of suspected Lyme borreliosis in Denmark: survey of patients seen in general practice.

Dessau RB, Bangsborg JM, Ejlertsen T, Skarphedinsson S, Schønheyder HC.

Department of Clinical Microbiology, Næstved Hospital, Region Zealand, Næstved, Denmark.

BACKGROUND: Serological testing for Lyme borreliosis (LB) is frequently requested by general practitioners for patients with a wide variety of symptoms.

METHODS: A survey was performed in order to characterize test utilization and clinical features of patients investigated for serum antibodies to Borrelia burgdorferi sensu lato. During one calendar year a questionnaire was sent to the general practitioners who had ordered LB serology from patients in three Danish counties (population 1.5 million inhabitants). Testing was done with a commercial ELISA assay with purified flagella antigen from a Danish strain of B. afzelii.

RESULTS: A total of 4,664 patients were tested. The IgM and IgG seropositivity rates were 9.2% and 3.3%, respectively. Questionnaires from 2,643 (57%) patients were available for analysis. Erythema migrans (EM) was suspected in 38% of patients, Lyme arthritis/disseminated disease in 23% and early neuroborreliosis in 13%. Age 0-15 years and suspected EM were significant predictors of IgM seropositivity, whereas suspected acrodermatitis was a predictor of IgG seropositivity. LB was suspected in 646 patients with arthritis, but only 2.3% were IgG seropositive. This is comparable to the level of seropositivity in the background population indicating that Lyme arthritis is a rare entity in Denmark, and the low pretest probability should alert general practitioners to the possibility of false positive LB serology. Significant predictors for treating the patient were a reported tick bite and suspected EM.

CONCLUSIONS: A detailed description of the utilization of serology for Lyme borreliosis with rates of seropositivity according to clinical symptoms is presented. Low rates of seropositivity in certain patient groups indicate a low pretest probability and there is a notable risk of false positive results. 38% of all patients tested were suspected of EM, although this is not a recommended indication due to a low sensitivity of serological testing.

PMCID: PMC2990752 PMID: 21040576 [PubMed - indexed for MEDLINE]

8. J Clin Pathol. 2010 Aug;63(8):719-21. Epub 2010 Jun 30.

More specific bands in the IgG western blot in sera from Scottish patients with suspected Lyme borreliosis.

Evans R, Mavin S, McDonagh S, Chatterton JM, Milner R, Ho-Yen DO.

Microbiology Department, Raigmore Hospital, Inverness, UK.

AIMS: To identify further Western blot bands that may be specific in the

diagnosis of Lyme borreliosis.

METHODS: The Borrelia burgdorferi antibody profiles of 270 western blot positive patients and 241 western blot negative patients from 2008 were examined.

RESULTS: 27 different non-specific bands were detected in both groups. Six of 27 (22%) of the non-specific bands were detected significantly more in the western blot positive patients compared to the western blot negative patients (20 kDa, p<0.0001; 28 kDa, p<0.002; 36 kDa, p<0.002; 37 kDa, p<0.007; 48 kDa, p<0.023; 56 kDa, p<0.028; two-tailed F test).

CONCLUSION: Results suggest that the 20, 28 and 48 kDa bands should be regarded as specific.

PMID: 20595179 [PubMed - indexed for MEDLINE]

9. APMIS. 2010 Apr;118(4):313-23.

Simultaneous use of serum IgG and IgM for risk scoring of suspected early Lyme borreliosis: graphical and bivariate analyses.

Dessau RB, Ejlertsen T, Hilden J.

Department of Clinical Microbiology, Naestved Hospital, Region Zealand, Naestved, Denmark.

The laboratory diagnosis of early disseminated Lyme borreliosis (LB) rests on IgM and IgG antibodies in serum. The purpose of this study was to refine the statistical interpretation of IgM and IgG by combining the diagnostic evidence provided by the two immunoglobulins and exploiting the whole range of the quantitative variation in test values. ELISA assays based on purified flagella antigen were performed on sera from 815 healthy Danish blood donors as negative controls and 117 consecutive patients with confirmed neuroborreliosis (NB). A logistic regression model combining the standardized units of the IgM and IgG ELISA assays was constructed and the resulting disease risks graphically evaluated by receiver operating characteristic and 'predictiveness' curves. The combined model improves the discrimination between NB patients and blood donors. Hence, it is possible to report a predicted risk of disease graded for each individual patient, as is theoretically preferable. The predictiveness curve, when adapted to the local pretest probability of LB, allows high-risk and low-risk thresholds to be defined instead of cut-offs based on the laboratory characteristics only, and it allows the extent of under- and over-treatment to be assessed. It is shown that an example patient with low ELISA results in IgM and IgG, considered negative by the conventional cut-off, has a relatively high risk of belonging to the truly diseased population and a low risk of being false positive. Using a 20% high-risk threshold for advising the clinician to consider treatment, the sensitivity of the assay is increased from 76% to 85%, while the specificity is maintained at around 95%.

PMID: 20402677 [PubMed - indexed for MEDLINE]

10. Clin Vaccine Immunol. 2010 Jun;17(6):904-9. Epub 2010 Apr 14.

Rapid, simple, quantitative, and highly sensitive antibody detection for lyme disease.

Burbelo PD, Issa AT, Ching KH, Cohen JI, Iadarola MJ, Marques A.

Neurobiology and Pain Therapeutics Section, 49 Convent Drive, Building 49, Room 1C20, NIDCR, NIH, Bethesda, MD 20892-4410, USA.

There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease, an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n = 46) were used for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera, the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic performance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n = 139) used for validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%; P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity (95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B. burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.

PMCID: PMC2884422 PMID: 20392886 [PubMed - indexed for MEDLINE]

11. MMW Fortschr Med. 2009 Apr 30;151(18):8.

[New biomarker discovered. Will the diagnosis of neuroborreliosis soon be easier (interview by Maria Weiss)?].

[Article in German]

Rupprecht T.

PMID: 19769063 [PubMed - indexed for MEDLINE]

12. Rev Assoc Med Bras. 2009 Mar-Apr;55(2):139-44.

[Epidemiological characteristics of Lyme-like disease in children].

[Article in Portuguese]

Passos SD, Gazeta RE, Latorre Mdo R, Durigon EL, Gauditano G, Yoshinari NH.

Depto de Pediatria, Faculdade de Medicina de Jundiaí, SP.

BACKGROUND: To determine the prevalence, age distribution, seasonality and clinical characteristics of Lyme-simile disease in Brazilians less than 15 years of age.

METHODS. From July, 1998 to November, 2000, a cross-sectional study was conducted in 333 patients with skin rash and fever. Paired blood samples were collected for identification of the pathogens. Only 193 samples which were negative for other pathogens (Parvovirus B19 Human, Herpesvirus 6 Human, Measles, Rubella, Dengue, Scarlet fever and Enterovirus), were tested for borreliosis by Enzyme-Linked Immunosorbent Assay and Western-blotting. Other clinical, socioeconomic, demographic and climatic variables were studied.

RESULTS: Prevalence of the disease was 6.2%(12/193). Of the variables studied, there was predominance in: <6 years old (83.2%); females (66.7%); being from the city of Franco da Rocha (58.3 %); and a summer/fall seasonality. The duration of care was 4 days. Signs and symptoms with statistical significance were itching; absence of lip notch and ocular pain; irritability and good clinical condition. Other clinical data presented were: pruritus (90%), irritability (80%) and fever (38 masculineC) (58.3%) with a duration of 1 to 3 days. Erythema was maculo-papular (40%), urticaria-like (25%) and scarlatiniform (16.7%), occurring predominately on the trunk (60%). There were no primary clinical evidences of Lyme-simile disease in the patients under study. The sensitivity and specificity of the clinical diagnosis as opposed to the laboratory diagnosis was zero. There was no initial clinical suspicion of the disease in the 10 cases studied and followed up for two years that showed no evidence of cardiologic or neurological complications. This is the first study of Lyme-simile in Brazilian children.

CONCLUSION: Prevalence of Lyme-simile disease was low, and it was not remembered at the initial diagnosis of those with skin rash. However, practical knowledge is necessary, demanding increased medical attention.

PMID: 19488647 [PubMed - indexed for MEDLINE]

13. Diagn Microbiol Infect Dis. 2009 Jul;64(3):347-9. Epub 2009 Apr 18.

The C6 Lyme antibody test has low sensitivity for antibody detection in cerebrospinal fluid.

Vermeersch P, Resseler S, Nackers E, Lagrou K.

University Hospitals Leuven, Belgium.

Our aim was to evaluate the performance of the commercial Immunetics C6 Lyme ELISA assay as a screening assay for anti-Borrelia burgdorferi antibodies in cerebrospinal fluid (CSF). Sensitivity of C6 ELISA was determined in 28 consecutive patients who were diagnosed with neuroborreliosis and had evidence for intrathecal antibody synthesis on immunoblot analysis. The presence of additional bands in CSF or of bands with a stronger intensity in CSF compared with serum was considered evidence of intrathecal synthesis. All 28 patients tested borderline or positive with C6 ELISA on serum. Of the 28 CSF samples, 17 (61%) and 19 (68%) tested positive with C6 ELISA using a threshold of 0.9 and 0.5 (optical density/cutoff). The C6 Lyme ELISA tested has a low sensitivity for antibody detection in cerebrospinal fluid compared with immunoblot analysis.

PMID: 19376674 [PubMed - indexed for MEDLINE]

14. Pediatr Infect Dis J. 2009 May;28(5):394-7.

Prediction of Lyme meningitis based on a logistic regression model using clinical and cerebrospinal fluid analysis: a European study.

Tuerlinckx D, Bodart E, Jamart J, Glupczynski Y.

Département de Pédiatrie, Universitaires de Mont-Godinne, Yvoir, Belgium.

BACKGROUND: A prediction model based on clinical and cerebrospinal fluid (CSF) analysis has been proposed for the differentiation of Lyme meningitis (LM) from non-Lyme aseptic meningitis (NLAM) in the United States. No similar model has ever been proposed for European patients. The objective of our study was to develop a prediction model to differentiate LM from NLAM based on clinical and CSF biologic data.

METHODS: The medical charts of all children older than 2 years of age admitted to our hospital from 1996 through 2006 with a definite diagnosis of LM were analyzed and compared retrospectively with those having a diagnosis of NLAM. Chart review included the duration of symptoms, the presence of cranial neuropathy, and CSF analysis.

RESULTS: A total of 93 patients were included (LM: 26 patients; NLAM: 67 patients) in the study. Patients with LM had statistically more frequent cranial neuropathy (73% vs. 4%), displayed a longer duration of symptoms before admission (8.8 vs. 1.8 days), had a higher CSF protein (71 vs. 38 mg/d), and had a lower percentage of neutrophil cells in the CSF (3.4% vs. 51%) than patients with NLAM. A predicted probability was derived from these 4 variables. At a cutoff point of >0.432, the model had a negative predictive value of 100% and a positive predictive value of 92.3%, with a sensitivity of 100% and a specificity of 97%.

CONCLUSIONS: We report the first European prediction model for LM. Owing to its high negative predictive value, this model may assist physicians in managing aseptic meningitis (AM) while awaiting serologic tests, especially in Lyme endemic regions.

PMID: 19295463 [PubMed - indexed for MEDLINE]

15. Rev Med Chir Soc Med Nat Iasi. 2008 Apr-Jun;112(2):496-501.

[Results of etiologic diagnosis in clinical syndrome consistent with acute and chronic borreliosis].

[Article in Romanian]

Persecă T, Feder A, Molnar GB.

Institutul de Sănătate Publică, Prof. Dr. Iuliu Moldovan" Cluj Napoca.

Borreliosis is a multisystem infection, which in the absence of adequate diagnosis and clinical management, may develop towards various clinical forms of chronic pathology. Due to the heterogeneity of clinical manifestations it is known under more names: erythema migrans, Lyme disease, neuroborreliosis etc.

MATERIAL AND METHOD: Taking into account the present interest and the weight in pathology of syndromes consistent with the suspicion of a Borrelia spp. infection, since 2002 we applied in current practice the investigation of this etiology. There have been investigated 481 subjects, clinically suspected of Borrelia spp. infection that had historical risk of tick bite and cases of serous meningitis, after exclusion of usual etiology. Tests were performed on ELISA kits with standardised immunoreagents and recently, for result validation, on Western immunoblot kits (WB).

RESULTS: Our results revealed the Borrelia etiology in 32% of cases (27.96-36.29% CI = 95%) at the screening, value expressed by the persistent positivity of the specific immunoglobulins (Ig) IgM (80.5%) and IgM+IgG (19.5%). Historic infection, represented exclusively by IgG positivity, was present in 8.6% (5.87-11.98% CI = 95%) from the cases that were negative for IgM (68%, 63.71-72.04%, CI = 95%). This weight is superposable with the results obtained in investigating a comparable sample of healthy individuals (193 subjects with 6.74% historical IgG, 3.79-10.96%, CI = 95%). Based on these results, it can be considered that ELISA procedure is useful and of reliable prognosis value for screening the Borrelia spp. etiology, the next step, taking into account the higher sensitivity of WB, being WB procedure which is useful for confirmation of ELISA positive cases and for treatment efficiency surveillance. The results prove that Borrelia spp. infections are a public health issue, which due to the diversity of clinical manifestations and diagnosis difficulties need repeated and complex laboratory investigations.

PMID: 19295026 [PubMed - indexed for MEDLINE]

16. Clin Vaccine Immunol. 2008 Dec;15(12):1796-804. Epub 2008 Oct 22.

Evaluation of the recombinant VlsE-based liaison chemiluminescence immunoassay for detection of Borrelia burgdorferi and diagnosis of Lyme disease.

Ledue TB, Collins MF, Young J, Schriefer ME.

Foundation for Blood Research, 8 Science Park Road, Scarborough, ME 04074, USA.

Recent efforts to improve the serologic diagnosis of Lyme disease have included the use of a synthetic peptide (C6) that reproduces the sequence of invariable region 6 of VlsE, the variable surface antigen of Borrelia burgdorferi. In the present study, the diagnostic performance of DiaSorin's recombinant VlsE-based chemiluminescence immunoassay in 1,947 human serum samples was evaluated. Sensitivity was determined using two serum panels from the CDC. For panel I, we observed sensitivities of 68.4% and 75.6% for subjects with early, localized (n=19) or disseminated (n=41) disease, respectively. For panel II, we observed sensitivities of 61.5% and 100% for subjects with early (n=26) or late-stage (n=11) disease, respectively. We observed a specificity of 99.5% for healthy donors (n=600) living either in regions of the United States where the disease is endemic or in regions where it is not endemic. Overall, specificity among 207 potentially cross-reactive sera from subjects who had other spirochetal infections, nonspirochetal infections including bacterial and viral infections, or autoimmune or neurologic disease; who were positive for rheumatoid factor or anti-mouse antibodies; or who had been previously vaccinated for Lyme disease was 93.7%. In a direct comparison of 1,038 prospectively collected samples for Lyme disease testing we observed a relative sensitivity of 70%, a relative specificity of 99.1%, and an overall agreement of 97.1% between the DiaSorin recombinant VlsE chemiluminescence immunoassay and the Immunetics peptide-based C6 enzyme-linked immunosorbent assay.

PMCID: PMC2593167 PMID: 18945880 [PubMed - indexed for MEDLINE]

17. Clin Infect Dis. 2008 Oct 1;47(7):910-4.

Effect of Borrelia burgdorferi genotype on the sensitivity of C6 and 2-tier testing in North American patients with culture-confirmed Lyme disease.

Wormser GP, Liveris D, Hanincová K, Brisson D, Ludin S, Stracuzzi VJ, Embers ME, Philipp MT, Levin A, Aguero-Rosenfeld M, Schwartz I.

Division of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, NY 10595, USA.

BACKGROUND: A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species. This is a particular concern for the C6 test, which is based on reactivity to a single peptide.

METHODS: C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from >158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC). The sonicated whole cell-based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31.

RESULTS: The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) (P<.001); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type-defined genotypes. The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study (P=.07); this relationship was not observed with C6 testing.

CONCLUSIONS: Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease.

PMCID: PMC2773679 PMID: 18724824 [PubMed - indexed for MEDLINE]

18. Pediatr Infect Dis J. 2008 Jul;27(7):605-12.

Improved laboratory diagnostics of Lyme neuroborreliosis in children by detection of antibodies to new antigens in cerebrospinal fluid.

Skogman BH, Croner S, Forsberg P, Ernerudh J, Lahdenne P, Sillanpää H, Seppälä I.

Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköoping University, Sweden.

BACKGROUND: Laboratory diagnostics in Lyme neuroborreliosis need improvement. We hereby investigate 4 new recombinant or peptide Borrelia antigens in cerebrospinal fluid in children with neuroborreliosis to evaluate their performance as diagnostic antigens.

METHODS: An enzyme-linked immunosorbent assay was used to detect IgG antibodies to recombinant decorin binding protein A (DbpA), BBK32, outer surface protein C (OspC), and the invariable region 6 peptide (IR6). The recombinant antigens originated from 3 pathogenic subspecies; Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto. Cerebrospinal fluid and serum from children with clinical features indicative for neuroborreliosis (n = 57) were analyzed. Classification of patients was based on clinical symptoms and laboratory findings. Controls were children with other neurologic diseases (n = 20) and adult patients with no proven infection (n = 16).

RESULTS: Sensitivity for DbpA was 82%, for BBK32 70%, for OspC 58% and for IR6 70%. Specificities were 94%, 100%, 97%, and 97%, respectively. No single antigen was superior. When new antigens were combined in a panel, sensitivity was 80% and specificity 100%. The reference flagella antigen showed a sensitivity of 60% and a specificity of 100%. Over all, the B. garinii related antigens dominated.

CONCLUSIONS: Recombinant DbpA and BBK32 as well as the peptide antigen IR6 perform well in laboratory diagnostics of neuroborreliosis in children. New antigens seem to improve diagnostic performance when compared with the routine flagella antigen. If different antigens are combined in a panel to cover the antigenic diversity, sensitivity improves further and a specificity of 100% can be achieve.

PMID: 18536620 [PubMed - indexed for MEDLINE]

19. Clin Infect Dis. 2008 Jul 15;47(2):196-7.

Editorial commentary: laboratory testing for Lyme disease: time for a change?

Weinstein A.

Department of Medicine and Division of Rheumatology, Georgetown University School of Medicine,, Washington, DC, USA.

Comment on Clin Infect Dis. 2008 Jul 15;47(2):188-95.

PMID: 18532894 [PubMed - indexed for MEDLINE]

20. Clin Infect Dis. 2008 Jul 15;47(2):188-95.

Prospective study of serologic tests for lyme disease.

Steere AC, McHugh G, Damle N, Sikand VK.

Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Comment in Clin Infect Dis. 2008 Oct 15;47(8):1111-2; author reply 1112-3. Clin Infect Dis. 2008 Jul 15;47(2):196-7.

BACKGROUND: Tests to determine serum antibody levels-the 2-tier sonicate immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and Western blot method or the IgG of the variable major protein-like sequence-expressed (VlsE) sixth invariant region (C6) peptide ELISA method-are the major tests available for support of the diagnosis of Lyme disease. However, these tests have not been assessed prospectively.

METHODS: We used these tests prospectively to determine serologic responses in 134 patients with various manifestations of Lyme disease, 89 patients with other illnesses (with or without a history of Lyme disease), and 136 healthy subjects from areas of endemicity and areas in which the infection was not endemic.

RESULTS: With 2-tier tests and the C6 peptide ELISA, only approximately one-third of 76 patients with erythema migrans had results that were positive for IgM or IgG seroreactivity with Borrelia burgdorferi in acute-phase samples. During convalescence, 3-4 weeks later, almost two-thirds of patients had seroreactivity with the spirochete B. burgdorferi. The frequencies of seroreactivity were significantly greater among patients with spirochetal dissemination than they were among those who lacked evidence of disseminated disease. Of the 44 patients with Lyme disease who had neurologic, heart, or joint involvement, all had positive C6 peptide ELISA results, 42 had IgG responses with 2-tier tests, and 2 patients with facial palsy had only IgM responses. However, among the control groups, the IgG Western blot was slightly more specific than the C6 peptide ELISA. The differences between the 2 test systems (2-tier testing and C6 peptide ELISA) with respect to sensitivity and specificity were not statistically significant.

CONCLUSIONS: Except in patients with erythema migrans, both test systems were sensitive for support of the diagnosis of Lyme disease. However, with current methods, 2-tier testing was associated with slightly better specificity.

PMID: 18532885 [PubMed - indexed for MEDLINE]

21. Mayo Clin Proc. 2008 May;83(5):566-71.

Diagnosis and treatment of Lyme disease.

Bratton RL, Whiteside JW, Hovan MJ, Engle RL, Edwards FD.

Department of Family Medicine, Mayo Clinic, 13400 E Shea Blvd, Scottsdale, AZ 85259, USA.

Lyme disease is the most common tick-borne disease in the United States. This review details the risk factors, clinical presentation, treatment, and prophylaxis for the disease. Information was obtained from a search of the PubMed and MEDLINE databases (keyword: Lyme disease) for articles published from August 31, 1997, through September 1, 2007. Approximately 20,000 cases of Lyme disease are reported annually. Residents of the coastal Northeast, northwest California, and the Great Lakes region are at highest risk. Children and those spending extended time outdoors in wooded areas are also at increased risk. The disease is transmitted to humans through the bite of the Ixodes tick (Ixodes scapularis and Ixodes pacificus). Typically, the tick must feed for at least 36 hours for transmission of the causative bacterium, Borrelia burgdorferi, to occur. Each of the 3 stages of the disease is associated with specific clinical features: early localized infection, with erythema migrans, fever, malaise, fatigue, headache, myalgias, and arthralgias; early disseminated infection (occurring days to weeks later), with neurologic, musculoskeletal, or cardiovascular symptoms and multiple erythema migrans lesions; and late disseminated infection, with intermittent swelling and pain of 1 or more joints (especially knees). Neurologic manifestations (neuropathy or encephalopathy) may occur. Diagnosis is usually made clinically. Treatment is accomplished with doxycycline or amoxicillin; cefuroxime axetil or erythromycin can be used as an alternative. Late or severe disease requires intravenous ceftriaxone or penicillin G. Single-dose doxycycline (200 mg orally) can be used as prophylaxis in selected patients. Preventive measures should be emphasized to patients to help reduce risk.

PMID: 18452688 [PubMed - indexed for MEDLINE]

22. Clin Vaccine Immunol. 2008 Jun;15(6):981-5. Epub 2008 Apr 16.

Significantly improved accuracy of diagnosis of early Lyme disease by peptide enzyme-linked immunosorbent assay based on the borreliacidal antibody epitope of Borrelia burgdorferi OspC.

Jobe DA, Lovrich SD, Asp KE, Mathiason MA, Albrecht SE, Schell RF, Callister SM.

Microbiology Research Laboratory, Gundersen Lutheran Medical Center, 1300 Badger Street, La Crosse, WI 54601, USA.

Highly specific borreliacidal antibodies are induced by infection with Borrelia burgdorferi, and the immunodominant response during early Lyme disease is specific for an epitope within the 7 amino acids nearest the C terminus of OspC. We evaluated the ability of an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (OspC7) that matched the region to detect the response and compared the sensitivity during early Lyme disease to that for an FDA-approved Western blot. When the optical density value was adjusted to 98% specificity based on the results from testing normal or uncharacterized sera (n = 236) or sera from patients with blood factors or illnesses that commonly produce antibodies that cross-react with B. burgdorferi antigens (n = 77), 115 (73%) of 157 sera from patients likely to have early Lyme disease were positive for immunoglobulin M (IgM) antibodies and 17 (11%) also had IgG antibodies. In addition, the IgM ELISA reactivities and the titers of antibodies detected by a flow cytometric borreliacidal antibody test correlated closely (r = 0.646). Moreover, the IgM ELISA was significantly more sensitive (P < 0.001) than the Western blot procedure. The findings therefore confirmed that the peptide IgM ELISA detected OspC borreliacidal antibodies and provided strong evidence that the test can eliminate the necessity for confirming early Lyme disease by a supplementary test such as Western blotting.

PMCID: PMC2446615 PMID: 18417666 [PubMed - indexed for MEDLINE]

23. FEMS Microbiol Lett. 2008 Jun;283(1):30-5. Epub 2008 Apr 1.

Detection of Borrelia burgdorferi sensu lato DNA by PCR in serum of patients with clinical symptoms of Lyme borreliosis.

Santino I, Berlutti F, Pantanella F, Sessa R, del Piano M.

Department of Public Health Sciences, Sapienza University of Rome, Rome, Italy.

Lyme borreliosis is a disease caused by spirochaetes belonging to the genospecies complex Borrelia burgdorferi sensu lato (s.l.) transmitted by Ixodes ticks. At present, serology remains the main diagnostic tool for laboratory diagnosis of Lyme borreliosis. Recently, the PCR technique has been applied for diagnosis of B. burgdorferi s.l., but, until now, a reliable, easy-to-perform and sensitive method has not been described. Here we present a new PCR-based method for the detection of both B. burgdorferi s.l. and Borrelia genospecies DNAs in serum samples collected from patients showing Lyme disease symptoms. Of 265 serum samples of patients included in this study, 7.5% were positive, 1.9% was borderline and 90.6% were negative for antibodies against B. burgdorferi by enzyme-linked immunosorbent assay and Western blotting. The B. burgdorferi s.l. 16S rRNA gene was detected by PCR in all serum-positive and in two borderline samples. None of the serum-negative samples nor serum samples collected from healthy subjects gave positive PCR reactions. Of PCR-positive serum samples, 50% gave a positive reaction for Borrelia afzelii, 18% for Borrelia garinii and 23% for two Borrelia species. Two samples (9%) were not identified to species level. The new protocol could be considered to be reliable as neither false-positive nor false-negative reactions were recorded, and to be sensitive as it detects DNA from one bacterial cell.

PMID: 18384365 [PubMed - indexed for MEDLINE]

24. Mol Gen Mikrobiol Virusol. 2008;(1):18-22.

[Isolation of the recombinant proteins OspC and the fragment FlaA (F-FLAA) from the western-Siberian Borrelia garinii NT29 isolates and the study of their immunochemical properties].

[Article in Russian]

Karavaev VS, Ivanov ID, Riabchenko AV, Beklemishev AB.

The structural proteins OspC and FlaA of the Lyme disease (LD) agent are known to be the basic antigens, which induce the humoral immune response at the initial stage of the disease. The goal of this work was to obtain the recombinant OspC and a fragment of the FlaA protein (f-FlaA) from the Western Siberian Borrelia garinii NT29 isolates and to assess the possibility of their use for the LD diagnosis. Encoding regions of the OspC and f-FlaB genes were amplified using PCR inserted in the pREB expressive vectors and cloned in the E. coli str. BL21 and C-600, respectively. The recombinant OspC and f-FlaA proteins were purified using affinity chromatography on Ni-NTA-sepharose 6A, and their ability to bind serum antibody of patients with Lyme disease was tested using western-blot and ELISA methods. The results of the analyses suggest that these proteins can be considered as promising components for elaboration of diagnostic tests for LD. The prototype of the ELISA diagnostic test was designed on the basis of the OspC and f-FlaA recombinant antigens. This test provides satisfactory parameters of diagnostic specificity (70.0%) and sensitivity (85.0%).

PMID: 18368777 [PubMed - indexed for MEDLINE]

25. Vector Borne Zoonotic Dis. 2008 Jun;8(3):381-90.

Evaluation of recombinant line immunoblot for detection of Lyme disease in Slovakia: comparison with two other immunoassays.

Lencáková D, Fingerle V, Stefancíková A, Schulte-Spechtel U, Petko B, Schréter I, Wilske B.

Department of Natural Foci Diseases, Parasitological Institute, Slovak Academy of Sciences, Kosice, Slovakia.

In the present study the sensitivity and the specificity of three serological tests (enzyme-linked immunosorbent assay [ELISA], indirect fluorescent antibody test [IFA], and recombinant line immunoblot) were compared by examining 74 sera from patients diagnosed with Lyme disease in Eastern Slovakia. In addition, the reactivity to each of the recombinant proteins in the immunoblot was examined in order to evaluate their diagnostic value. Generally, the immunoblot (93.2%) and the ELISA (90.5%) were significantly more sensitive than the IFA (64.9%; df = 1; p < or = 0.001). Correlation between results of the ELISA, IFA, and immunoblot for IgM or IgG, when two tests were always compared, one to the other, ranged from r(s) = 0.673 to r(s) = 0.905. In the immunoblot, the highest sensitivity was observed in DbpA and VlsE proteins (76.9% and 84.6%, respectively) in IgG testing of the sera from the patient group of Lyme arthritis. VlsE proteins, together with OspC proteins, were also shown to be useful for IgM antibody detection in erythema migrans patients (up to 44.4% and 53.7% sensitivity, respectively). Our results indicate that both the ELISA and the recombinant immunoblot test were more satisfactory for seroconfirmation of Lyme disease than IFA. Moreover, the reseach confirmed diagnostic value of the in-vivo expressed proteins (VlsE and DbpA), which might have the potential to play an important role in improving whole-cell antigen-based testing.

PMID: 18279004 [PubMed - indexed for MEDLINE]

26. Med Pr. 2007;58(5):439-47.

[Diagnostics of Lyme disease].

[Article in Polish]

Gasiorowski J, Witecka-Knysz E, Knysz B, Gerber H, Gładysz A.

Katedra i Klinika Chorób Zakaźnych, Chorób Watroby i Nabytych Niedoborów Odpornościowych Akademia Medyczna, Wrocław.

Although many years have passed since Borrelia burgdorferi was first identified, advances in understanding biology and clinical course of infection made and new diagnostic procedures developed, Lyme disease is still difficult to diagnose. Therefore, it is often wrongly diagnosed and unnecessarily treated. In this paper we analyzed the latest data on Lyme disease diagnostic methods, paying much attention to their limitations and correct interpretation of results. In routine diagnosis of this diseases, indirect tests, based on the detection of specific IgM and IgG antibodies, are most useful. The Lyme disease diagnosis should begin with screening tests, which are highly sensitive but not specific enough and sometimes yield false positive results, then all positive results should be verified by confirmation tests, which allow to distinguish between true positive results and healthy individuals with false positive ones.

PMID: 18274096 [PubMed - indexed for MEDLINE]

27. Ann Agric Environ Med. 2007 Dec;14(2):209-13.

Molecular and serological diagnosis of Borrelia burgdorferi infection among patients with diagnosed Erythema migrans.

Kondrusik M, Grygorczuk S, Skotarczak B, Wodecka B, Rymaszewska A, Pancewicz S, Zajkowska J, Swierzbińska R, Hermanowska-Szpakowicz T.

Department of Infectious Diseases and Neuroinfections, Medical University, Zurawia 14, 15-540 Białystok, Poland.

The aim of the study was to assess the frequency of Borrelia burgdorferi DNA detection in the blood and urine of patients diagnosed with erythema migrans, and compare the results of PCR-based methods with ELISA methodology. The latter was used to detect serum antibodies against Borrelia burgdorferi of the IgM and IgG classes, before and after antibiotic therapy. The study included 86 patients hospitalized in the Department of Infectious Diseases and Neuroinfections in the Medical Academy in Białystok, diagnosed with the erythema migrans phase of Lyme borreliosis. Examinations were carried out twice: the first at the moment of diagnosis (Trial 1), the second after 4 weeks of antibiotic therapy. The study showed that antibiotic therapy in the early phase of borreliosis does not decrease the sensitivity of PCR and that after 4 weeks of therapy (Trial 2), spirochete DNA is still detectable in most patients (45/86). There was no correlation between detectability of spirochete DNA and the presence of antibodies against B. burgdorferi s.l. (assessed by ELISA) during the course of erythema migrans. The largest percentage of positive results in the detection of B. burgdorferi s.l. DNA was observed in patients who simultaneously possessed IgM and IgG antibodies against B. burgdorferi, while the lowest percentage of PCR positive results was among patients with only IgM antibodies.

PMID: 18247452 [PubMed - indexed for MEDLINE]

28. Bratisl Lek Listy. 2007;108(9):399-402.

Antibodies to Borrelia burgdorferi in erythema migrans patients.

Trnovcova M, Bazovska S, Svecova D.

Department of Epidemiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia.

Determination of antibodies against Borrelia burgdorferi has supporting value in the diagnose of Lyme disease. The purpose of this study was to determine the production of antibodies in a defined group of patients.MATERIAL AND

METHODS: The study analysed antibodies in the group of 25 patients with erythema migrans. For the detection of antibodies Immunofluorescence methods, ELISA and Western blot were used.

RESULTS: The detection of antibodies by Imunoflorescence methods proved positivity of the titre 1:256 in 14 patients and 9 patients were borderline, with the titre 1:128. Majority of the antibodies detected were of IgM class. The ELISA IgM test found positive reaction in 12 patients and 2 borderline results. IgG antibodies were found significantly more often by ELISA test than by Immunofluorescence. The Western blotting results were IgM positive in 9 patients, 6 were borderline. Only 5 patients have positive IgM results in all tests (Immunofluorescence, ELISA and at least one positive result out of three IgM Western blots). DISCUSSION AND CONCLUSION: The tests which detect Borrelia burgdorferi antibodies are not standardized. They have variable sensitivity and specificity and their standardization is complicated with respect to great heterogenicity of Borrelia burgdorferi strains circulating in individual regions of Europe. The high specificity of antibodies to individual borrelia antigens are presently pointing towards the need to use, when in diagnostic confusion, more tests, which could detect antibodies also to other borrelia antigens (Tab. 3, Ref. 14). Full Text (Free, PDF)

PMID: 18225477 [PubMed - indexed for MEDLINE]

29. Pol Merkur Lekarski. 2007 Aug;23(134):95-9.

[Comparison of test with antigen VlsE (C6) with tests with recombinant antigens in patients with Lyme borreliosis].

[Article in Polish]

Zajkowska JM, Kondrusik M, Pancewicz SA, Grygorczuk S, Jamiołkowski J, Stalewska J.

Akademia Medyczna w Białymstoku, Klinika Chorób Zakaźnych i Neuroinfekcji.

In diagnosis of Lyme borreliosis classic, recombinant antigens are used. Introduced recombinant antigen VlsE increases hope to improve sensibility of the tests.AIM OF THE STUDY: Serological results detecting antibodies against Borrelia burgdorferi, recombinant antigens in both classes and test with VlsE were compared. MATERIAL AND

METHODS: Quick ELISA C6 Borrelia Assay Kit (U.S.A.) for both classes simultaneusly were compared with two tests composed of recombinant antigens Borrelia IgG recombinant, Borrelia IgM recombinant (Biomedica, Austria)separate for IgM i IgG antibodies. Patients with borreliosis as eryhtema migrans were evaluated (n=36) and group with chronic Lyme arthritis (n=68).

RESULTS: Statistical coincidence between results of test ELISA C6 and presence of antibodies IgM (p < 0.0001) and IgG (p < 0.0001) obtained in tests based on recombinant antigens in group erytema migrans. In group Lyme arthritis coincidence (p < 0.0002), with IgM antibodies was stated.

CONCLUSIONS: Test ELISA C6 (with protein VlsE-C6) in group of patients with Lyme borreliosis had statistically essential predictive values of presence antibodies in IgM i IgG class, stated in tests performed separately for IgM and IgG classes with recombinant antigens. Tests based on conservative part of protein VlsE (C6), detecting antibodies in both classes are very promising in diagnosis of early stages Lyme borreliosis.

PMID: 18044336 [PubMed - indexed for MEDLINE]

30. Int J Med Microbiol. 2008 Jul;298(5-6):493-504. Epub 2007 Sep 24.

Comparison of different Borrelia burgdorferi sensu lato strains for detection of immune response in patients with erythema migrans.

Merljak Skocir L, Ruzić-Sabljić E, Maraspin-Carman V, Lotric-Furlan S, Logar M, Strle F.

Institute for Public Health, Vipavska cesta 13, 5000 Nova Gorica, Slovenia.

The aim of the present study was to establish which combination of serological method and Borrelia strain used as an antigen would provide the most appropriate demonstration of borrelial infection in patients with eythema migrans residing in Slovenia. Four different strains were chosen as antigens: two strains of B. afzelii and two strains of B. garinii which differed in their expression of the outer proteins OspA, OspB and OspC. Each individual strain was used as antigen in immunofluorescence test (IFT), enzyme-linked immunosorbent assay (EIA) with whole borrelial cells, and EIA with ultrasonicated borrelial cells. With these 12 different tests, 100 samples were examined for the presence of specific IgM and IgG antibodies: 50 sera of blood donors and 50 sera of patients with erythema migrans. The latter were further subdivided into skin culture-positive and -negative subgroups. A commercial Western blot (WB) test was performed for 26 sera of the control group and 25 sera of patients with erythema migrans. The four different methods had distinct specificity and sensitivity. The most specific approaches were IFT (100% for IgM and 90-92% for IgG) and the WB test (100% for IgM and 73% for IgG), followed by EIA with whole borrelial cells (80-98% for IgM and 76-84% for IgG) and EIA with ultrasonicated borrelial cells (76-94% for IgM and 72-80% for IgG). The sensitivity levels of all these tests were low. The most sensitive were EIA tests with whole borrelial cells (28-36% for IgM and 32-42% for IgG) followed by EIA with ultrasonicated borrelial cells (22-32% for IgM and 24-36% for IgG), the WB test (16% for IgM and 32% for IgG) and IFT (0-2% for IgM and 14-20% for IgG). The following methods gave significant differences between patients and negative controls in detecting IgM antibodies: EIA with whole borrelial cells with both B. afzelii antigens and with antigen B. garinii that expressed OspA and OspC, EIA with ultrasonicated borrelial cells with antigen B. afzelii that expressed OspA, OspB and OspC. In detecting IgG antibodies, significant differences were observed between EIA with whole borrelial cells and with antigen B. afzelii that expressed OspA and OspB. Borreliae were isolated from the skin of 34/50 (68%) patients with erythema migrans: two strains failed to grow, while 26/32 (81%) strains were identified as B. afzelii, 5/32 (16%) as B. garinii and 1/32 (3%) as B. burgdorferi sensu stricto. No statistically significant differences in serologic test results between culture-positive and -negative patients with erythema migrans were found.

PMID: 17892971 [PubMed - indexed for MEDLINE]

31. Diagn Microbiol Infect Dis. 2007 Dec;59(4):355-63. Epub 2007 Sep 20.

Antigen biochips verify and extend the scope of antibody detection in Lyme borreliosis.

Du W, Ma X, Nyman D, Povlsen K, Akguen N, Schneider EM.

Section Experimental Anesthesiology, University Clinic Ulm, D-89075 Ulm, Germany.

The antibody response of serum IgM and IgG of patients with neuroborreliosis and erythema migrans of Lyme borreliosis (LB) was examined against a 41-kDa flagellar antigen and an 8-mer synthetic OspC8 peptide (VAESPKKP) derived from the C-terminus of outer surface protein C (OspC) from Borrelia garinii. We developed a streptavidin-modified biochip-based immunodiagnosis and compared it with conventional methods such as enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). The diagnostic sensitivity of the coated biochips was demonstrated to be identical, and the results of conventional assays such as ELISA and WB were confirmed. Flagellar antigens lead to better diagnosis because of a higher discriminative value. By contrast, OspC8, a peptide derived from the outer surface antigen, is less sensitive to identify immunity in LB. The inferior antigenicity of OspC8 may be due to epitope masking. Overall, this system is open to simultaneously analyze a larger family of peptides differing in length. Thus, an array approach is generally more advantageous to extend the pattern of antigens to be tested for antigenicity in LB. Serial analysis during ongoing disease may be valuable to learn more about the course of the disease and intermittent reactivation of infection. Protein biochip as a potential substitution of ELISA and WB method offers the opportunity to study serum immunity in a multiplicity of patients simultaneously.

PMID: 17888607 [PubMed - indexed for MEDLINE]

32. Neurology. 2007 Sep 4;69(10):953-8.

Relevance of the antibody index to diagnose Lyme neuroborreliosis among seropositive patients.

Blanc F, Jaulhac B, Fleury M, de Seze J, de Martino SJ, Remy V, Blaison G, Hansmann Y, Christmann D, Tranchant C.

Department of Neurology, University Hospital of Strasbourg, Louis Pasteur University, Strasbourg, France.

Comment in Neurology. 2008 Jul 8;71(2):150; author reply 150-1. Neurology. 2007 Sep 4;69(10):949-50.

BACKGROUND: No consensual criteria exist to diagnose neuroborreliosis. The intrathecal anti-Borrelia antibody index (AI) is a necessary criterion to diagnose neuroborreliosis in Europe, but not in the United States. Previous studies to determine the diagnostic value of the AI found a sensitivity ranging from 55% to 80%. However, these studies included only typical clinical cases of meningitis or meningoradiculitis, and none had a control group with CSF anti-Borrelia antibodies.

METHODS: We studied a sample of 123 consecutive patients with clinical signs of neurologic involvement and CSF anti-Borrelia antibodies. We determined the AI for all patients and a final diagnosis was made. Patients were then divided into three groups (neuroborreliosis, possible neuroborreliosis, control).

RESULTS: Thirty of the 40 patients with neuroborreliosis had a positive AI (AI sensitivity = 75%). Two of the 74 patients with another neurologic diagnosis had a positive AI (AI specificity = 97%). CONCLUSION: The antibody index has a very good specificity but only moderate sensitivity. Given the lack of consensual criteria for neuroborreliosis and the absence of a "gold standard" diagnostic test, we propose pragmatic diagnostic criteria for neuroborreliosis, namely the presence of four of the following five items: no past history of neuroborreliosis, positive CSF ELISA serology, positive anti-Borrelia antibody index, favorable outcome after specific antibiotic treatment, and no differential diagnosis. These new criteria will need to be tested in a larger, prospective cohort.

PMID: 17785663 [PubMed - indexed for MEDLINE]

33. Eur J Clin Microbiol Infect Dis. 2007 Jul;26(7):517-9.

Comparison of an automated Borrelia indirect chemiluminescent immunoassay (CLIA) with a VlsE/C6 ELISA and Immunoblot.

Riesbeck K, Hammas B.

Medical Microbiology, Department of Laboratory Medicine, Malmö University Hospital, Lund University, 205 02, Malmö, Sweden.

PMID: 17558488 [PubMed - indexed for MEDLINE]

34. Clin Vaccine Immunol. 2007 Jul;14(7):875-9. Epub 2007 May 30.

Epitope length, genospecies dependency, and serum panel effect in the IR6 enzyme-linked immunosorbent assay for detection of antibodies to Borrelia burgdorferi.

Gomes-Solecki MJ, Meirelles L, Glass J, Dattwyler RJ.

New York Medical College, BSB Room 308, Valhalla, NY 10595, USA.

In the absence of erythema migrans, the basis for diagnosis of Lyme disease is the demonstration of an antibody response against Borrelia burgdorferi in an appropriate clinical setting. The C6 enzyme-linked immunosorbent assay, based on the IR6 region of VlsE, has become widely used in both the United States and Europe. We mapped the antigenic epitopes of IR6 to a shorter sequence that is equivalent in sensitivity and specificity to the full-length IR6 25-residue peptide. In addition, we observed significant differences in sensitivity between serum panels (60 to 100%), indicating that the selection of the serum panels can shape the apparent overall sensitivity of the assay. Contrary to prior reports, the assay sensitivity is greater when the IR6 peptide is derived from the sequence of the same infecting Borrelia genospecies. Using our North American panels and the two panels obtained from European Lyme disease patients, we determined that the IR6 assay that is based on a single genospecies of Borrelia spp. is not optimal for use as a universal diagnostic assay for Lyme disease.

PMCID: PMC1951069 PMID: 17538122 [PubMed - indexed for MEDLINE]

35. Med Mal Infect. 2007 Jul-Aug;37(7-8):496-506. Epub 2007 May 23.

[Role of biological assays in the diagnosis of Lyme borreliosis presentations. What are the techniques and which are currently available?].

[Article in French]

De Martino SJ.

Laboratoire associé au CNR Borrelia, laboratoire de bactériologie, hôpitaux universitaires de Strasbourg, 3, rue Koeberlé, 67000 Strasbourg, France.

The biological diagnosis of Borrelia burgdorferi sensu lato infection is usually made by antibody detection in patient sera. Thus, serological testing (Elisa, immunoblotting) is essential for a biological diagnosis. Specific antibody detection is usually done in serum and CSF of patients suspected of Lyme borreliosis. Laboratories must follow European recommendations to validate these assays in routine practice. Antibody detection lacks sensitivity in the early cutaneous phase of the infection. Therefore, serological testing is not recommended for the diagnosis of erythema migrans. The interpretation of serology must take into account the variability of Elisa sensitivity and specificity and the lack of standardization for Western-blotting in Europe. Besides these indirect diagnosis techniques, there is also direct detection of spirochetes by culture or by in vitro DNA amplification but these require adequate samples. These molecular tests must not be performed routinely, but only for specific clinical situations and in specialized laboratories only.

PMID: 17512148 [PubMed - indexed for MEDLINE]

36. Int J Med Microbiol. 2007 Feb;297(1):45-52. Epub 2007 Jan 17.

Immune responses to borrelial VlsE IR6 peptide variants.

Sillanpää H, Lahdenne P, Sarvas H, Arnez M, Steere A, Peltomaa M, Seppälä I.

Department of Bacteriology and Immunology, Haartman Institute, FI-00014 Helsinki, Finland.

Laboratory confirmation of Lyme borreliosis (LB) relies mainly on the demonstration of anti-borrelial antibodies. In recent studies, a novel VlsE protein IR(6) peptide-based assay has been introduced. Our aim was to evaluate the IR(6) peptides from three Borrelia burgdorferi sensu lato genospecies in the serodiagnosis of European and North American patients. Five VlsE protein IR(6) peptide variants representing sequences from B. burgdorferi sensu stricto, B. garinii, and B. afzelii were used as antigens in both IgG and IgM enzyme-linked immunosorbent assays (ELISA). Serum antibodies of 187 patients at different stages of LB from Europe and the United States were evaluated for serodiagnosis. For comparison samples were tested with one of the commercial IR(6) ELISAs. Three B. afzelii IR(6) variant peptides revealed antibodies that were concordant with each other. B. burgdorferi sensu stricto peptide antibodies mostly paralleled B. afzelii peptide antibodies, and positive values were also obtained in the majority of European sera. For several sera, B. garinii IR(6) peptide antibodies were discordant to B. afzelii peptide antibodies. The commercial IR(6) peptide antibody assay (C6 ELISA) results correlated better with B. burgdorferi sensu stricto IR(6) than with B. garinii IR(6) peptide IgG results, especially in sera from patients with facial palsy. Thus, antibody specificity to IR(6) peptides may vary according to the infecting Borrelia species. In some manifestations of the disease, C6 ELISA may not cover all LB cases. Evidently, the methodological aspects in ELISA design for peptide antibody measurements are important as well as the amino acids sequence of the antigen.

PMID: 17234451 [PubMed - indexed for MEDLINE]

37. Eur J Clin Microbiol Infect Dis. 2007 Jan;26(1):37-42.

C6 peptide ELISA test in the serodiagnosis of Lyme borreliosis in Sweden.

Tjernberg I, Krüger G, Eliasson I.

Stensö Primary Healthcare Center, Lasarettsvägen, Kalmar, Sweden.

The aim of this study was to evaluate the synthetic C6 peptide test as a first-line test in a two-tiered scheme for Borrelia serology in a clinically well-characterized population of patients with Lyme borreliosis in Kalmar County, Sweden. The study population consisted of a prospective group (n = 200), a control group (n = 255), and a retrospective group (n = 29). The test panel consisted of the Immunetics Quick ELISA C6 Borrelia assay kit (Immunetics, Cambridge, MA, USA), the Virotech Borrelia burgdorferi ELISA (Genzyme Virotech, Rüsselsheim, Germany), and the Liaison Borrelia CLIA (DiaSorin, Saluggia, Vercelli, Italy). Seroprevalence among 200 healthy blood donors was significantly lower in the C6 test (8%) compared to the Virotech ELISA (14%) and the Liaison CLIA (12%). In convalescent sera (2-3 months and 6 months post infection) from 158 patients with erythema migrans, the seropositivity in the C6 test was also significantly lower compared to both the Virotech ELISA and the Liaison CLIA. Serosensitivity in the acute phase of erythema migrans and other clinical manifestations of borreliosis did not differ significantly between the C6 test and the Virotech ELISA or the Liaison CLIA. Overall, a positive C6 test seems to correlate well with acute borreliosis. Cross-reactivity was lower in the C6 test in sera positive for Epstein-Barr virus infection as compared to the Virotech ELISA. This study supports the use of the C6 test as a screening test for borreliosis, in endemic areas.

PMID: 17180348 [PubMed - indexed for MEDLINE]

38. Wien Klin Wochenschr. 2006 Nov;118(21-22):677-81.

Paradigm Burgenland: risk of Borrelia burgdorferi sensu lato infection indicated by variable seroprevalence rates in hunters.

Cetin E, Sotoudeh M, Auer H, Stanek G.

Clinical Institute for Hygiene and Medical Microbiology, Medical University of Vienna, Vienna, Austria.

BACKGROUND: This study concerns the prevalence of antibodies against B. burgdorferi sensu lato as an indicator of previous borrelial infection among hunters, a group of occupationally exposed persons. In order to define associated risk factors and preparing data for future comparisons, a study was performed in the eight districts of Burgenland, the most eastern state of Austria.

METHODS: Blood samples of 1214 men (median age 51 years, range 18 to 89 years) and 39 women (median age 44 years, range 21 to 69 years) were collected during autumn 2002 and winter 2003. Demographic data regarding age, sex, profession, residence, duration of employment (hunters), hunting-ground, animals in the environment, and history of tick bite were obtained by a questionnaire. A two-step testing strategy was used in which sera were screened for antibodies against B. burgdorferi sensu lato by a commercially available recombinant enzyme-linked immunosorbent assay (Biotest Anti-Borrelia IgG ELISA; Biotest AG, Dreieich, Germany). Reactive sera were then subjected to immuno blot testing (recomBlot Borrelia; Mikrogen, Munich, Germany) for confirmation of specificity.

RESULTS: A total of 673 (54%) sera tested positive for IgG antibodies to Borrelia burgdorferi sensu lato: 663 (55%) men and 10 (26%) women. Seropositivity was clearly related to age and duration of hunting activity; it was 33% among persons younger than 29 years and 83% in those older than 70 years. Further, there was also a difference in the distribution of seroprevalence within the districts; the highest was found in hunters from the most southern district of Burgenland, Jennersdorf, (69%) and the lowest was noticed in the most northern district, Neusiedl (39%).

CONCLUSIONS: We found an overall seroprevalence of 54% in asymptomatic hunters of Burgenland. Infectious risk exists in the entire state but the prevalence rate differs in the various districts indicating a variable risk which peaks in the south. The nearly linear increase of seroprevalence with age and duration of hunting activity reflects repeated tick exposure.

PMID: 17160606 [PubMed - indexed for MEDLINE]

39. Wien Klin Wochenschr. 2006 Nov;118(21-22):625-33.

Lyme borreliosis in 2005, 30 years after initial observations in Lyme Connecticut.

Steere AC.

Center for Immunology and Inflammatory Diseases, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

Nearly 100 years ago, Afzelius described a patient with an expanding skin lesion, called erythema migrans, which is now known to be the initial skin manifestation of Lyme borreliosis. Approximately 70 years later, in 1976, epidemiologic evaluation of a cluster of children with arthritis in Lyme, Connecticut led to a complete description of the infection. During the subsequent years, investigators in a number of countries have made remarkable strides in the elucidation of this tick-borne spirochetal infection. The purpose of this review is to discuss the current status of Lyme borreliosis, including areas in which knowledge of the infection is still incomplete.

PMID: 17160599 [PubMed - indexed for MEDLINE]

40. Clin Vaccine Immunol. 2007 Jan;14(1):90-3. Epub 2006 Nov 15.

Borrelia burgdorferi spirochetes that harbor only a portion of the lp28-1 plasmid elicit antibody responses detectable with the C6 test for Lyme disease.

Embers ME, Wormser GP, Schwartz I, Martin DS, Philipp MT.

Tulane National Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Road, Covington, LA 70433, USA.

Detection of antibody to C6, a peptide that reproduces the sequence of the sixth invariable region within the central domain of the VlsE protein of Borrelia burgdorferi, is used currently for the serologic diagnosis of Lyme disease in humans. B. burgdorferi isolates taken from infected humans can be categorized into specific genetic subtypes (designated RST1, -2, and -3) by restriction fragment length polymorphisms in the 16S to 23S rRNA spacer sequence. Many of these, usually categorized as RST2, retain only segments of the linear plasmid lp28-1, which encodes VlsE. The VlsE genetic region is retained, but altered expression of this molecule could affect diagnosis by the C6 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients infected with each of the three genotypes and from mice infected with three RST2 isolates were tested with the C6 ELISA. Such isolates elicited marked C6 responses in infected mice. The sensitivity of C6 antibody detection in patients infected with RST2 spirochetes was statistically indistinguishable from detection of RST1 and RST3 infections. These findings demonstrate that diagnosis by C6 ELISA remains effective for infection with all B. burgdorferi genotypes, including those with incomplete lp28-1 plasmids.

PMCID: PMC1797709 PMID: 17108288 [PubMed - indexed for MEDLINE]

41. J Med Microbiol. 2006 Nov;55(Pt 11):1499-504.

Antigenicity of borrelial protein BBK32 fragments in early Lyme borreliosis.

Lahdenne P, Sarvas H, Kajanus R, Eholuoto M, Sillanpää H, Seppälä I.

Hospital for Children and Adolescents, University of Helsinki, Stenbäckinkatu 11, FIN-00290 Helsinki, Finland.

Recombinantly produced borrelial BBK32 protein fragments originating from Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii were evaluated as antigens in the serology of Lyme borreliosis (LB). In ELISA, a mid-portion hydrophilic fragment reacted with LB patient sera. Of the 23 patients with culture- or PCR-positive erythema migrans (EM), 43 % at diagnosis and 52 % at convalescence were positive for at least one Borrelia species-specific variant BBK32 fragment antigen. In parallel ELISAs with BBK32 whole proteins from the three borrelial subspecies as antigens, 17 % at diagnosis and 26 % at convalescence were positive. These results suggest that BBK32 protein fragments may improve the early IgG serology of LB compared to the BBK32 whole protein.

PMID: 17030908 [PubMed - indexed for MEDLINE]

42. J Clin Microbiol. 2006 Oct;44(10):3778-80.

Identification of Borrelia burgdorferi ribosomal protein L25 by the phage surface display method and evaluation of the protein's value for serodiagnosis.

Mueller M, Bunk S, Diterich I, Weichel M, Rauter C, Hassler D, Hermann C, Crameri R, Hartung T.

University of Konstanz, Biochemical Pharmacology, 78457 Konstanz, Germany.

The phage surface display technique was used to identify Borrelia burgdorferi antigens. By affinity selection with immunoglobulin G from pooled sera of six Lyme borreliosis (LB) patients, the ribosomal protein L25 was identified. The diagnostic value of L25 was investigated by an enzyme-linked immunosorbent assay, using sera from 80 LB patients and 75 controls, and the use of the protein resulted in a specificity of 99% and a 23% sensitivity, which qualify L25 as a useful antigen when combined with others.

PMCID: PMC1594769 PMID: 17021109 [PubMed - indexed for MEDLINE]

43. Przegl Epidemiol. 2006;60 Suppl 1:177-85.

[Western-blot with VLSE protein and "in vivo" antigens in Lyme borreliosis diagnosis].

[Article in Polish]

Zajkowska J, Kondrusik M, Pancewicz S, Grygorczuk S, Swierzbińska R, Hermanowska-Szpakowicz T, Czeczuga A, Sienkiewicz I.

Klinika Chorób Zakainych i Neuroinfekcji AM w Białymstoku.

The aim of the study was the evaluation of the efficiency of Western blot (EcoLine) test detecting simoultanous presence of IgM and IgG antibodies against B. burgdorferi in diagnosis of early and late stage of Lyme borreliosis. The comparison of results achieved by performing test Western-blot, ELISA (based on recombinant antigens of three genospecies of Borrelia) and EIA (based on antigens of one B. burgdorferi genospecies). The tests Western blot: EcoLine (Virotech) with antygens "in vivo", ELISA Borrelia IgM, IgG recombinant (Biomedica), EIA: B. b. ss. IgG, EIA B. garinii IgG, EIA B. afzelii IgG (TestLine) were used. Results showed efficacy of detecting IgM, IgG antibodies against VlsE simultanously and IgG antibodies against "in vivo" antigens in diagnosis of early stages of Lyme disease when atypical picture skin lessions arise diagnostic doubts and in discerning early and late stage of disease. The EIA tests based on one B. burgdoreferi genospecies seem less effective in comparison to ELISA tests based on 3 genospecies antigens.

PMID: 16909799 [PubMed - indexed for MEDLINE]

44. Przegl Epidemiol. 2006;60 Suppl 1:171-6.

[Comparison of two types of diagnostic test detecting antibodies against Borrelia burgdorferi: EIA based on one genospecies antigens and ELISA based on recombinant antigens].

[Article in Polish]

Zajkowska J, Kondrusik M, Grygorczuk S, Pancewicz S, Swierzbińska R, Sienkiewic I, Hermanowska-Szpakowicz T.

Klinika Chorób Zakaźnych i Neuroinfekcji, Akademii Medycznej w Białymstoku.

The aim of the study was to compare the results of ELISA diagnostic kit detecting antibodies against B. burgdorferi based on combination of three genospecies recombinant and EIA kits based on one of three genospecies. Sera of 351 forest workers were evaluated with ELISA kit (Recombinant antigen, IgG). Seropositive samples were tested with EIA kits based on B. burgdorferi s.s., B. garinii, B. afzelii antigens. Diagnostic kits based on combination of antigens of three genospecies more often detect antibodies against B. burgdorferi and are more usefull as screening tests, in comparison with those based on one genospecies. Among diagnostic kits based on one genospecies, the most sesnitive in detection of antibodies against B. burgdorferi are those based on B. afzelii.

PMID: 16909798 [PubMed - indexed for MEDLINE]

45. New Microbiol. 2006 Apr;29(2):139-41.

Evaluation of the C6 enzyme-linked immunoadsorbent assay for the serodiagnosis of Lyme borreliosis in north-eastern Italy.

Cinco M, Murgia R.

Laboratorio Spirochete, Dipartimento di Scienze Biomediche, Università di Trieste, Italy.

A novel antigen preparation--the synthetic C6 peptide, a conserved portion of the variable VlsE antigens of Borrelia burgdorferi--has been evaluated for serodiagnosis of Lyme borreliosis (LB) by an ELISA procedure. Serum specimens were from early and late LB patients, all resident in an endemic area in north-eastern Italy. The specificity of the test approached the 100% and sensitivity was in the order of 63% (early LB) and 100% (late LB); this performance is superior to the preceding generation of Lyme disease tests.

PMID: 16841555 [PubMed - indexed for MEDLINE]

46. Clin Microbiol Infect. 2006 May;12(5):496-7.

VlsE C6 peptide and IgG ELISA antibody analysis for clinical diagnosis of Lyme borreliosis in an endemic area.

Nyman D, Willén L, Jansson C, Carlsson SA, Granlund H, Wahlberg P.

PMID: 16643532 [PubMed - indexed for MEDLINE]

47. Ther Umsch. 2005 Nov;62(11):737-44.

[Diagnostic tests of Lyme borreliosis].

[Article in German]

Steffen I, Hirsch HH.

Institut für Medizinische Mikrobiologie, Universität Basel, Basel.

The diversity of Borrelia burgdorferi subspecies (sensu lato) in Europe does not allow to simply adopt the US definitions and tests which were established for B. burgdorferi sensu stricto. Routine detection of Borrelia-specific antibodies now includes specific antigens of the subspecies B. afzelii and B. garinii. Thus, the 2-step serology of screening by ELISA and confirmation by immunoblot remains the most important diagnostic test of lyme borreliosis. The detection of borrelia by PCR or culture is of lesser importance due to the limited sensitivity, despite of the high specificity. In clinical practice, the risk of exposure to ticks and the stage of the clinical presentation guide the choice of appropriate diagnostic procedures and their interpretation.

PMID: 16350536 [PubMed - indexed for MEDLINE]

48. Ann Med. 2005;37(8):568-79.

Epidemiology and diagnosis of Lyme borreliosis.

Wilske B.

Max von Pettenkofer-Institute, University of Munich, National Reference Center for Borreliae, Pettenkofer-Strasse 9a, D-80336 Munich, Germany.

The multisystem disease Lyme borreliosis is the most frequent tick-transmitted disease in the northern hemisphere. In Europe Lyme borreliosis is most frequent in Central Europe and Scandinavia (up to 155 cases per 100,000 individuals) and is caused by the species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. The recently detected genospecies A14S may also play a role in skin manifestations. Microbiological diagnosis in European patients must consider the heterogeneity of borreliae for development of diagnostic tools. According to guidelines of the USA and Germany, serological diagnosis should follow the principle of a two-step procedure (enzyme-linked immunosorbent assay (ELISA) as first step, if reactive; followed by immunoblot). The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens (p100, p58, p41i, VlsE, OspC, DbpA) including those expressed primarily in vivo (VlsE and DbpA) instead of whole cell lysates. VlsE is the most sensitive antigen for IgG antibody detection, OspC for IgM antibody detection. 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. Detection of the etiological agent by culture or polymerase chain reaction (PCR) should be confined to specific indications and specialized laboratories. Recommended specimens are skin biopsy specimens, cerebrospinal fluid (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 except when the duration of symptoms is shorter than 2 weeks (50% sensitivity). Methods which are not recommended or adequately documented for diagnosis are antigen tests on body fluids, PCR of urine, and lymphocyte transformation tests.

PMID: 16338759 [PubMed - indexed for MEDLINE]

49. Int Immunol. 2005 Dec;17(12):1631-7. Epub 2005 Nov 22.

Different B-cell populations are responsible for the peripheral and intrathecal antibody production in neuroborreliosis.

Lakos A, Ferenczi E, Komoly S, Granström M.

Center for Tick-borne Diseases, Visegrádi 14, H-1132 Budapest, Hungary.

The diagnosis of neuroborreliosis (NB)--a serious complication of Lyme disease--relies on demonstration of intrathecal borrelia antibody production. We hypothesized that if a qualitative difference between the cerebrospinal fluid and the serum immunoblot-banding patterns was observed, then the borrelia antibodies found in the CSF could not be the result of leakage of serum antibodies to the CSF due to blood-brain barrier damage, but rather had to be produced intrathecally. CSF/serum pairs from 69 NB patients and from 85 control patients with other neurological disorders were investigated. All samples were tested blindly by immunoblot and a commercial capture ELISA kit for NB. The concordance between the two methods was 85.7%. When using the other method as reference, the accuracy of the two assays in the two patient materials was similar: 80% for sensitivity and 95% for specificity. Four types of comparative immunoblot-banding patterns that reflected intrathecal borrelia antibody synthesis were distinguished. The study showed that a simple comparison between the immunoblot pattern of serum and CSF samples allows for a reliable diagnosis of NB by demonstration of intrathecal antibody production. This is the first study to show that a qualitative difference of the antibody response between the immune response of serum and CSF is a rule. The findings also imply that partly different B-cell populations are responsible for the antibody production in the blood and in the central nervous system. In addition, our observation provides possible implications for other infectious diseases with CNS involvement.

PMID: 16303786 [PubMed - indexed for MEDLINE]

50. Clin Diagn Lab Immunol. 2005 Sep;12(9):1069-74.

A decline in C6 antibody titer occurs in successfully treated patients with culture-confirmed early localized or early disseminated Lyme Borreliosis.

Philipp MT, Wormser GP, Marques AR, Bittker S, Martin DS, Nowakowski J, Dally LG.

Department of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, LA 70433, USA.

C(6), a Borrelia burgdorferi-derived peptide, is used as the antigen in the C(6)-Lyme disease diagnostic test. We assessed retrospectively whether a fourfold decrease or a decrease to a negative value in anti-C(6) antibody titer is positively correlated with a positive response to treatment in a sample of culture-confirmed patients with either early localized (single erythema migrans [EM]; n=93) or early disseminated (multiple EM; n=27) disease. All of these patients had been treated with antibiotics and were free of disease within 6 to 12 months of follow-up. Results show that a serum specimen taken at this time was either C(6) negative or had a >or=4-fold decrease in C(6) antibody titer with respect to a specimen taken at baseline (or during the early convalescent period if the baseline specimen was C(6) negative) for all of the multiple-EM patients (P<0.0001) and in 89% of the single-EM patients (P<0.0001). These results indicate that a decline in anti-C(6) antibody titer coincides with effective antimicrobial therapy in patients with early localized or early disseminated Lyme borreliosis.

PMCID: PMC1235797 PMID: 16148173 [PubMed - indexed for MEDLINE]

51. Acta Neurol Scand. 2005 Aug;112(2):97-102.

Relevance of immunological variables in neuroborreliosis and multiple sclerosis.

Bednárová J, Stourac P, Adam P.

Department of Clinical Microbiology, Faculty Hospital, Masaryk University, Brno, Czech Republic.

OBJECTIVES: The aims were to investigate the frequency of intrathecal synthesis of specific antibodies against measles (M), rubella (R) and varicella zoster (Z) viruses (MRZ reaction) as a diagnostic marker between multiple sclerosis (MS) and neuroborreliosis (NB) groups and to postulate the most typical cerebrospinal fluid (CSF) variables profile of these entities.

METHODS: Three cohorts of patients were investigated: MS (n = 42), NB (n = 27) and other neurological diseases (OND) (n = 15). Measles, rubella, varicella zoster and borrelia-specific IgG antibodies were measured by ELISA, Q(alb) (CSF/serum albumin ratio) as a marker of blood-CSF barrier function and specific antibody indices (AI) were calculated according to relevant formulae. IgG oligoclonal bands (OB) were detected by isoelectric focusing and immunoenzymatic staining.

RESULTS: Eighty-eight percent of MS patients had positive MRZ reaction and 26.2% had positive anti-borrelia AI. Eighty-nine percent of NB patients had positive anti-borrelia AI and two patients had individually anti-measles and rubella positive AI. MS-CSF variables profile included the presence of IgG OB in 81%, elevated Q(alb) in 31% and normal cell count in 66.7%. Of NB patients IgG OB were positive in 74%, elevated Q(alb) in 81.5% and normal cell count in 7.4%. CONCLUSION: MRZ reaction was proved as statistically significant marker in differential diagnosis between MS and NB. Typical CSF variables profile of these two entities is highly supportive, especially when MRZ is included.

PMID: 16008535 [PubMed - indexed for MEDLINE]

52. Clin Diagn Lab Immunol. 2005 Jul;12(7):801-7.

Use of Peptide library screening to detect a previously unknown linear diagnostic epitope: proof of principle by use of lyme disease sera.

Hamby CV, Llibre M, Utpat S, Wormser GP.

Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA.

Diagnostic peptides previously isolated from phage-displayed libraries by affinity selection with serum antibodies from patients with Lyme disease were found to give reproducible serum reactivity patterns when tested in two different enzyme-linked immunosorbent assay formats. In addition, the hypothetical possibility that peptides selected by this type of "epitope discovery" technique might identify the original antigens eliciting antibody responses was tested by searching for sequence similarities in bacterial protein databases. In support of this hypothesis, our search uncovered similarities between peptides representing two different sequence motifs and sequences in the VlsE and BBA61 antigens of Borrelia burgdorferi. Utilizing synthetic peptides, we verified that the sequence KAASKETPPALNK, located at the C terminus of the VlsE antigen, had the same reactivity pattern to sera from patients with extracutaneous Lyme disease as the diagnostic peptide SKEKPPSLNWPA, with which it shared a 7-amino-acid-residue match (consensus residues are underlined). A peptide with conservative mutations of five of the consensus residues was nonreactive, strongly suggesting that the VlsE sequence represents the epitope that originally elicited antibody responses in these patients. The diagnostic sensitivity of this new VlsE epitope was relatively low (30%) compared to that (100%) of the well-documented C6 diagnostic peptide of VlsE when tested in our small cohort of 10 patients with Lyme disease. Nonetheless, the identification of this previously unknown epitope serves as a proof of the principle of the hypothetical ability of "epitope discovery" techniques to detect specific microbial antigens with diagnostic relevance in infectious diseases.

PMCID: PMC1182200 PMID: 16002626 [PubMed - indexed for MEDLINE]

53. Folia Microbiol (Praha). 2005;50(1):31-9.

Improved method of detection and molecular typing of Borrelia burgdorferi sensu lato in clinical samples by polymerase chain reaction without DNA purification.

Rudenko N, Golovchenko M, Nemec J, Volkaert J, Mallátová N, Grubhoffer L.

Faculty of Biological Sciences, University of South Bohemia, 370 05 Ceské Budejovice, Czechia.

A simple assay by polymerase chain reaction was used for the of detection of Borrelia burgdorferi, causative agent of Lyme borreliosis (LB). It involves no DNA purification and is based on the amplification of a specific region of ospA gene of B. burgdorferi, followed by direct detection of the PCR product with SYBR Green I by agarose gel electrophoresis. The method was used to analyze samples from patients with LB diagnosis, with presumable infection with the LB spirochete, those with unclear clinical symptoms and after the course of an antibiotic treatment. Spirochetal DNA was detected by PCR even in contaminated samples in which B. burgdorferi was overgrown by fungi and other bacteria. Spirochetal DNA was detected and borrelia species was identified in cerebrospinal fluid of two patients hospitalized with the diagnosis "fever of unknown origin". Western blot and ELISA were negative in both cases. Total analysis of 94 samples from the hospital in Ceské Budejovice (South Bohemia, Czechia) showed infection with B. burgdorferi sensu stricto in 11% and B. garinii in 15% of cases. The highest prevalence was found for B. afzelii (43%). Co-infection was confirmed in 24 % of the analyzed symplex; 7% of samples that were B. burgdorferi sensu lato positive gave no results in DNA amplification with B. burgdorferi sensu stricto-, B. garinii- and B. afzelii-specific primers. The proposed reliable, rapid, unexpensive and specific technique could form the basis of laboratory tests for routine detection and identification of Lyme-disease spirochete in different samples.

PMID: 15954531 [PubMed - indexed for MEDLINE]

54. Antibiot Khimioter. 2004;49(11):12-5.

[Preparations for serodiagnosis of diseases due to causative agents of ixodes tick-borne borreliosis (Lyme disease). Communication 3. Comparative study of enzyme immunoassay test-systems for detection of IgM to Borrelia burgdorferi sensu lato].

[Article in Russian]

Manzeniuk IN, Vorob'eva MS, Kozarenko AA, Andreĭchuk IuV.

Three foreign and one Russian ELISA test-systems for detection of IgM to Borrelia burgdorferi sensu lato were comparatively studied with the use of the clinical material in an encoded experiment. In the foreign ELISA test-systems based on the use of native antigens of borrelia, cross reactions with sera from patients with syphilis, Epstein-Barr infection, cytomegalovirus infection and systemic lupus erythematosus were detected. The Russian recombinant ELISA test-system Borreliosis-ELISA-IgM showed high sensitivity and specificity. The simultaneous use of the test-systems for detecting IgG and IgM significantly increased the efficacy of diagnosis of early borreliosis.

PMID: 15945543 [PubMed - indexed for MEDLINE]

55. Enferm Infecc Microbiol Clin. 2005 Apr;23(4):232-40.

[Diseases produced by Borrelia].

[Article in Spanish]

Escudero-Nieto R, Guerrero-Espejo A.

Laboratorio de Espiroquetas y Patógenos Especiales, Servicio de Bacteriología, Centro Nacional de Microbiología, ISCIII. Majadahonda, Madrid, Spain.

Lyme borreliosis, caused by Borrelia burgdorferi sensu lato, is a multi-organ infection with dermatological, rheumatological, neurological, and cardiac manifestations. The main characteristic is a skin lesion, named erythema migrans. Relapsing fever, caused by numerous species of Borrelia, is characterized by a periodic cycle of acute and afebrile episodes. The serological diagnosis of these infections has limited value in sensitivity, specificity and predictive values. Lyme borreliosis is usually diagnosed by recognition of a characteristic clinical picture with serological confirmation, and the diagnosis of relapsing fever relies on direct observation of spirochetes in peripherical blood. The elected treatment is almost always tetracycline for the young or for adults but not for pregnant women, although betalactamic (such as penicillin or 3rd generation cephalosporin for the central nervous system) or macrolides are indicated in several situations. The prognosis, with adequate treatment, is good. In the majority of Spanish regions, due to the low incidence of these diseases, the prophylactic antimicrobial treatment after a tick bite is not indicated.

PMID: 15826549 [PubMed - indexed for MEDLINE]

56. New Microbiol. 2005 Jan;28(1):37-43.

Comparative evaluation of two enzyme linked immunosorbent assay methods and three Western Blot methods for the diagnosis of culture-confirmed early Lyme borreliosis in Italy.

Marangoni A, Sparacino M, Mondardini V, Cavrini F, Storni E, Donati M, Cevenini R, Sambri V.

Sezione di Microbiologia DMCSS, University of Bologna, Ospedale Policlinico S Orsola, Bologna, Italy.

This study investigated the onset and development of the immune response to Borrelia burgdorferi infection in 30 Italian patients with culture-confirmed Lyme Borreliosis in the stage of erythema migrans (EM). All patients received antimicrobial treatment when entering the study and were prospectively evaluated monthly for up to 30 days after enrolment. A total of 60 serially collected serum samples were tested by using two different commercial enzyme-linked immunosorbent assays (ELISAs): Anti-Borrelia plus VlsE ELISA, Euroimmun, and the synthetic peptide-based ELISA, Quick ELISA C6, Immunetics. Sixty-five potentially cross-reacting sera were also tested. Anti-Borrelia plus VlsE ELISA IgG was far more sensitive than Quick ELISA C6 (56.6% and 33.3%, respectively). Moreover, considering that 17 additional sera from the first bleeding group of Lyme disease patients were IgM positive when tested by Anti-Borrelia plus VlsE IgM, the sensitivity of Anti-Borrelia plus VlsE as a whole system rose to 85.0%. Nevertheless, due to the specificity values of Anti-Borrelia plus VlsE ELISA identified in this study (98.5% for IgG and 78.5% for IgM), the need of a confirmatory test for the diagnosis of Lyme disease remains. All the sera were also tested by two different commercial Western Blot (WB) assays: Euroline-WB against Borrelia, Euroimmun, and Qualicode B. burgdorferi WB, Immunetics, in comparison with a multispecies "home made" WB. Performances of the three WB methods for the detection of IgM were very similar. On the contrary, these WBs performed with different values of sensitivity and specificity when IgGs were evaluated. The most sensitive method was the "home-made" WB IgG (71.7%), followed by the Euroline-WB IgG against Borrelia (68.3%). Qualicode B. burgdorferi WB IgG demonstrated to be only 26.6% sensitive. Both "home-made" WB IgG and Qualicode B. burgdorferi WB IgG were 100% specific, whereas Euroline-WB IgG against Borrelia scored 12 cross-reacting samples as borderline, showing a specificity value of 80.0%.

PMID: 15782625 [PubMed - indexed for MEDLINE]

57. J Med Microbiol. 2005 Apr;54(Pt 4):361-7.

Comparative evaluation of three different ELISA methods for the diagnosis of early culture-confirmed Lyme disease in Italy.

Marangoni A, Sparacino M, Cavrini F, Storni E, Mondardini V, Sambri V, Cevenini R.

Sezione di Microbiologia - DMCSS, University of Bologna, St Orsola Hospital, via Massarenti 9, Bologna, Italy.

In this study the raising and development of the immune response to Borrelia burgdorferi infection in 45 Italian patients suffering from culture-confirmed Lyme borreliosis erythema migrans was investigated. A total of 95 serially collected serum samples were tested by using three different commercial ELISAs: recomWell Borrelia (Mikrogen), Enzygnost Borreliosis (DADE Behring) and Quick ELISA C6 Borrelia (Immunetics). The sensitivities of the ELISAs were as follows: Enzygnost Borreliosis IgM, 70.5 %; Quick ELISA C6 Borrelia, 62.1 %; recomWell Borrelia IgM, 55.7 %; recomWell Borrelia IgG, 57.9 %; and Enzygnost Borreliosis IgG, 36.8 %. In order to compare the specificity values of the three ELISAs, a panel of sera obtained from blood donors (210 samples coming from a non-endemic area and 24 samples from an endemic area) was tested, as well as sera from patients suffering from some of the most common biological conditions that could result in false-positive reactivity in Lyme disease serology (n = 40). RecomWell Borrelia IgG and recomWell Borrelia IgM were the most specific (97.1 % and 98.9 %, respectively), followed by Quick ELISA C6 Borrelia (96.7 %). Enzygnost Borreliosis IgG and IgM achieved 90.1 % and 92.3 % specificity, respectively. Sera that gave discrepant results when tested by the three ELISAs were further analysed by Western blotting.

PMID: 15770021 [PubMed - indexed for MEDLINE]

58. Przegl Epidemiol. 2004;58(3):451-8.

[Activity of lysosomal exoglycosidases in serum of patients with chronic borrelia arthritis].

[Article in Polish]

Wielgat P, Pancewicz S, Hermanowska-Szpakowicz T, Kondrusik M, Zajkowska J, Grygorczuk S, Popko J, Zwierz K.

Zakład Biochemii Farmaceutycznej AM w Białymstoku.

To estimate activities of lisosomal exoglycosidases in serum of patients with chronic borrelia arthritis. Study group consisted of 18 patients aged 18-72 years (x=46) hospitalized in Department of Infectious Diseases and Neuroinfections of Medical Academy in Białystok with diagnosis of chronic arthritis in course of borreliosis. Control consisted of 20 healthy volunteers (health services employees) aged 25-65 years (x=45), with no detectable anti-Borrelia burgdorferi antibodies in serum. In all borreliosis patients serum activity of: N-acetyl-beta-D-glucosaminidase (HEX), beta-galactosidase and alpha-mannosidase was measured before and after 4 weeks of doxycycline treatment. Results were analyzed with Statistica 6.0 software. P < 0.05 was considered statistically significant. HEX activity was significantly increased in serum of Lyme arthritis patients before treatment compared to controls. It decreased after 4-week treatment, remaining insignificantly higher than in controls. b-galactosidase and a-mannosidase activities in serum of Lyme arthritis patients were insignificantly higher than in controls and fell after treatment to the levels observed in control group. N-acetyl-beta-D-glucosaminidase (HEX) is sensitive enzymatic marker of Lyme arthritis. It may be used to monitor course of the disease and its efficiency of treatment.

PMID: 15730009 [PubMed - indexed for MEDLINE]

59. Antibiot Khimioter. 2004;49(8-9):25-8.

[Preparations for serodiagnosis of diseases due to causative agents of ixode tick-borne Borreliosis (Lyme disease). Communication 2. Comparative study of recombinant enzyme immunoassay test-systems (rELISA) for serological diagnosis of ixode tick-borne Borreliosis].

[Article in Russian]

Manzeniuk IN, Vorob'eva MS, Nikitiuk NM, Arumova EA, Anan'eva LP, Baranova SG, Viatkina TG, Masiago AV, Kozarenko AA, Andreĭchuk IuA.

One foreign and two Russian recombinant enzyme immunoassay test-systems were comparatively investigated under conditions of an encoded experiment. Sensitivity in the experiment with the Russian test-systems Borreliosis-ELISA-IgG and Lyme Best was 63.8 and 68.8% respectively. As for the test-system Borrelia IgG Recombinant, it was 47.5%. All the test-systems were highly specific (94.4 to 99.5%). The test-systems Lyme Best and Borrelia IgG Recombinant revealed partial cross reactions with sera from patients with systemic lupus erythematosus and leptospirosis.

PMID: 15727142 [PubMed - indexed for MEDLINE]

60. Antibiot Khimioter. 2004;49(6):10-4.

[Preparations for serodiagnosis of diseases due to causative agents of ixode tick-borne borreliosis (Lyme disease). Communication 1. Comparative study of the 1st generation enzyme immunoassay test-systems for detection of antibodies to Borrelia burkdorferi sensu lato].

[Article in Russian]

Manzeniuk IN, Vorob'eva MS, Arumova EA, Barabova EV, Evsegneev SI, Anan'eva LP.

Four foreign and one Russian 1st generation test-systems for detecting class G antibodies or summary antibodies to Borrelia burkdorferi sensu lato were comparitively investigated with the use of the clinical material under conditions of an encoded experiment. Cross reactions with sera from patients with syphilis, Epstein-Barr infection, cytomegalovirus infection and systemic lupus erythematosus were observed. The best specificity and sensitivity parameters were provided by the Enzygnost Borreliosis test-system.

PMID: 15628796 [PubMed - indexed for MEDLINE]

61. Clin Diagn Lab Immunol. 2004 Sep;11(5):924-9.

Comparison of Western immunoblotting and the C6 Lyme antibody test for laboratory detection of Lyme disease.

Mogilyansky E, Loa CC, Adelson ME, Mordechai E, Tilton RC.

Medical Diagnostic Laboratories, Mt. Laurel, NJ 08054, USA.

Three commercial Lyme disease Western immunoblotting (WB) kits and the C6 Borrelia burgdorferi (Lyme) enzyme-linked immunosorbent assay (ELISA) kit were compared using two commercially available performance panels from the Centers for Disease Control and Prevention (CDC) and Boston Biomedica (BBI). Combined, the panels consisted of 52 characterized specimens. Immunoglobulin G (IgG) sensitivity was similar for the three WB products. The BBI and Marblot WBs were more specific for IgG antibodies, while the Virablot was the most sensitive for IgM antibody. The BBI WB was 100% specific for IgM, while Marblot was 97% and Virablot was 77% specific for IgM. The C6 ELISA was found to be 100% sensitive. Four false-positive C6 results were identified in patients that had clinically and microbiologically confirmed Lyme disease but were not detected by the CDC reference methods. No one WB product showed overall superiority. The C6 ELISA shows promise as the first ELISA for Lyme disease that would not require a supplemental test such as a WB.

PMCID: PMC515278 PMID: 15358654 [PubMed - indexed for MEDLINE]

62. Otol Neurotol. 2004 Sep;25(5):838-41.

The VlsE (IR6) peptide ELISA in the serodiagnosis of lyme facial paralysis.

Peltomaa M, McHugh G, Steere AC.

Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

OBJECTIVE: Facial paralysis is a manifestation of early disseminated Lyme neuroborreliosis. In the current study, we compared the immunoglobulin G (IgG) VlsE (sixth invariant region) peptide enzyme-linked immunosorbent assay (ELISA) with the current two-tier approach of sonicate ELISA and Western blot in the serodiagnosis of Lyme facial paralysis. STUDY DESIGN: Retrospective. SETTING: Tertiary referral center. PATIENTS: Serum samples from 47 Lyme patients with facial paralysis and 86 control subjects were analyzed for IgG antibodies to VlsE peptide of Borrelia burgdorferi and for immunoglobulin M (IgM) and IgG antibodies to sonicate antigens of B. burgdorferi using the two-tier approach. INTERVENTION: Diagnostic. MAIN OUTCOME MEASURE: Serum IgG antibody responses to VlsE (IR6) peptide.

RESULTS: All 47 (100%) patients with facial paralysis and 4 (5%) of 86 controls had positive antibody responses to the VlsE peptide. In the two-tier test, 41 (87%) patients had positive IgM, 31 (66%) had positive IgG, and all 47 patients had positive IgM or IgG responses. Of the 86 control subjects, 2 (2%) had positive results with the two-tier test. Thus, the sensitivities of the VlsE and the two-tier tests were 100%; the specificity of the VlsE ELISA was 95% and the specificity of the two-tier test was 98%.

CONCLUSIONS: The VlsE peptide ELISA showed a high sensitivity and specificity in the serological diagnosis of Lyme facial paralysis, similar to the two-tier test. The principal advantage of the VlsE peptide ELISA is that it requires only one test rather than four tests. However, the specificity of the VlsE test may not be as high as that of the two-tier test.

PMID: 15354020 [PubMed - indexed for MEDLINE]

63. Clin Diagn Lab Immunol. 2004 May;11(3):458-62.

Dogs vaccinated with common Lyme disease vaccines do not respond to IR6, the conserved immunodominant region of the VlsE surface protein of Borrelia burgdorferi.

O'Connor TP, Esty KJ, Hanscom JL, Shields P, Philipp MT.

Department of Research and Development, IDEXX Laboratories, One IDEXX Dr., Westbrook, ME 04092, USA.

A 25-amino-acid synthetic peptide (C(6) peptide) derived from an immunodominant conserved region (designated IR(6)) of the VlsE protein of Borrelia burgdorferi has been identified and used to construct immunoenzyme-based diagnostic procedures. These procedures have excellent sensitivity and specificity. Previous reports have demonstrated the usefulness of the C(6) peptide as an antigen for the serodiagnosis of human and canine Lyme disease. Results indicated that assays based on the C(6) peptide were nonreactive to sera from vaccinated nonexposed animals. The purpose of the present study was to confirm these results in a controlled trial by testing sera from experimentally vaccinated dogs known to be uninfected. Nine specific-pathogen-free beagles were assigned to one of three vaccine groups, each containing three dogs. Each group received one of three commercial Lyme vaccines: RECOMBITEK Lyme (Merial), LymeVax (Fort Dodge Animal Health), and Galaxy Lyme (Schering-Plough Animal Health). Each animal was administered a total of five doses of vaccine over a period of 39 weeks. Serum samples were collected prior to vaccination and then on a weekly basis from weeks 3 to 18 and from weeks 33 to 43. Selected samples were tested by the immunofluorescence assay (IFA) and the Western blot (WB) assay using whole-cell B. burgdorferi antigen extracts, and the results were compared to those obtained with two immunoenzyme-based procedures formatted by using the C(6) peptide. Serum specimens from all animals were reactive to the IFA and WB assay at week 5 and became highly reactive following the administration of multiple doses of vaccine. All serum specimens were uniformly nonreactive in the C(6) peptide immunoenzyme procedures at all time points throughout the study.

PMCID: PMC404571 PMID: 15138170 [PubMed - indexed for MEDLINE]

64. J Infect Dis. 2004 May 15;189(10):1962; author reply 1962-4.

Cost-effectiveness of peptide-antigen immunoassays for Lyme disease.

Porwancher RB.

Comment on J Infect Dis. 2003 Apr 15;187(8):1187-99.

PMID: 15122535 [PubMed - indexed for MEDLINE]

65. Vector Borne Zoonotic Dis. 2003 Winter;3(4):215-27.

Diagnosis of lyme borreliosis in europe.

Wilske B.

Max von Pettenkofer Institute, University of Munich, National Reference Center for Borreliae, Pettenkofer-Stresse 9a, D 80336 Munich, Germany.

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.

PMID: 14733674 [PubMed - indexed for MEDLINE]

66. J Neurol. 2003 Nov;250(11):1318-27.

Diagnosis of Lyme neuroborreliosis with antibodies to recombinant proteins DbpA, BBK32, and OspC, and VlsE IR6 peptide.

Panelius J, Lahdenne P, Saxén H, Carlsson SA, Heikkilä T, Peltomaa M, Lauhio A, Seppälä I.

Haartman Institute, Dept. of Bacteriology and Immunology, University of Helsinki, 21, 00014, Helsinki, Finland.

Three recombinant antigens, decorin binding protein A (DbpA), BBK32, and outer surface protein C (OspC), and IR(6) peptide of borrelial VlsE protein, were evaluated for the diagnosis of neuroborreliosis (NB), using cerebrospinal fluid (CSF) and serum samples from 89 patients. Their performances in enzyme-linked immunosorbent assay (ELISA) were compared with that of commercial flagella antigen. IgG ELISAs were performed with three variants of each recombinant antigen originating from Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii, and with the IR(6) peptide. IgM antibodies were analysed against OspC and flagella. Of the patients whose CSF contained elevated anti-flagella IgG antibodies, 93% were positive for at least three of the new antigens. Of those with negative or borderline CSF anti-flagella antibodies, 51% were positive for three new antigens. Antibodies to BBK32 were detectable mainly in early disease. Antibodies to DbpA and IR(6) were observed in early and late NB. The use of the new antigens at presentation of the disease improved the laboratory diagnosis of NB. In IgG ELISAs, the diagnostic sensitivity of assays with the new antigens was between 75 and 88%, but was only 52% with the flagella antigen. The discriminatory power between patient and control samples appeared better in the CSF than in the serum. We suggest that assessment of CSF antibodies to at least two antigens, using either flagella and one of the new antigens or two of the new antigens, would improve the current diagnostic yield of NB.

PMID: 14648148 [PubMed - indexed for MEDLINE]

67. APMIS. 2003 Nov;111(11):1053-9.

Improved serodiagnosis of early Lyme borreliosis: immunoblot with local Borrelia afzelii strain.

Jovicić VLj, Grego EM, Lako BL, Ristović BM, Lepsanović ZA, Stajković NT.

Institute of Public Health of Serbia Dr Milan Jovanović Batut, Department for Microbiology, Belgrade, Serbia and Montenegro.

To improve the serodiagnosis of Lyme borreliosis (LB) the performances of four tests were evaluated. An indirect immunofluorescent assay based on Borrelia burgdorferi s.s., enzyme-linked immunosorbent assay (ELISA) based on local isolates of Borrelia afzelii and B. burgdorferi s.s., and immunoblot (IB) of B. afzelii were prepared. The serum panels contained 214 serum samples: control group (n=120) and patients at different stages of LB (n=94). The specificity of IB was 96%, of in-house ELISA 93%, and of IFA 89%. In early LB the sensitivity of IFA was 36%, ELISA 67%, and IB 93%. In late-stage LB the sensitivity was: 72% for IFA, 80% for ELISA, and 94% for IB. Comparison of in-house and Behring ELISA showed that the sensitivity of the serological assay could be increased when the test was based on local Borrelia strains. IgM and IgG antibodies from sera of patients with early and late LB most frequently demonstrated reactivity to OspC. The other significant proteins in early LB were: p39, p41 in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB: p39, p41 in IgM IB, and p83/100, Osp17, p21 and p43 in IgG IB. Using IB based on local B. afzelii isolates improves the serodiagnosis of early LB in our geographical region.

PMID: 14629271 [PubMed - indexed for MEDLINE]

68. Diagn Microbiol Infect Dis. 2003 Sep;47(1):321-9.

Utility of a commercial immunoblot kit (BAG-Borrelia blot) in the diagnosis of the preliminary stages of Lyme disease.

Hernández-Novoa B, Orduña A, Bratos MA, Eiros JM, Fernández JM, Gutiérrez MP, Alonso PA, Mantecón MA, Almaraz A, Oteo JA, Rodríguez-Torres A.

Departamento de Microbiología, Hospital Universitario de Valladolid, Facultad de Medicina, Spain.

The aim of this study was to evaluate the usefulness of a commercial immunoblot (IgG and IgM BAG-Borrelia blot) in the serologic diagnosis of the early stages of Lyme disease. A total of 42 sera from patients with Lyme disease (24 patients with localized early stage (LES) and 18 patients with disseminated early stage (DES)) and 129 sera from patients with non-Lyme diseases (specificity control sera) were studied. IgG anti-p41 from Borrelia burgdorferi s.l. was present in 95.2% of patients followed by anti-p41/I PBi (16.7%), anti-p100 (9.5%) and anti-OspA (9.5%). IgM anti-p41 was present in 66.7% of patients, p41/iPBi (54.8%) and OspC (33.3%). IgM against p100, OspA and OspC were more frequent in DES patients (16.7%, 27.8% and 44.4%) than in LES patients (0.0%, 4.2% and 25.0%). In 4.8% of the cases no IgG bands were present and in 26.2% no IgM bands were present. With the exception of isolated p41 bands (59.5%), no band pattern exceeded 17%. Using manufacturer's instructions, test sensitivity in diagnosis of the early stage of Lyme disease is 61.9%, specificity 98.4% and positive and negative predictive values 92.8% and 88.8% respectively. Applying the EUCALB 5, 6 or 7 rules sensitivity increased to 73.8% although specificity decreased to 89.9%. Of the 129 specific control sera, 41.8% presented IgG anti-p41 and 10.8% IgM anti-p41. Patients with non-Lyme diseases that presented more IgG and IgM bands were those patients with syphilis (88.2%), patients with anti-HIV antibodies (57.8%) and patients with anti-nuclear antibodies (ANA) (52.3%).

PMID: 12967745 [PubMed - indexed for MEDLINE]

69. Lancet Infect Dis. 2003 Aug;3(8):489-500.

Lyme borreliosis.

Hengge UR, Tannapfel A, Tyring SK, Erbel R, Arendt G, Ruzicka T.

Department of Dermatology, Düsseldorf, Germany.

Erratum in Lancet Infect Dis. 2003 Dec;3(12):815.

Comment in Lancet Infect Dis. 2003 Nov;3(11):684. Lancet Infect Dis. 2004 Oct;4(10):603-4. Lancet Infect Dis. 2004 Apr;4(4):197-8; discussion 198-9.

Lyme borreliosis is a multi-organ infection caused by spirochetes of the Borrelia burgdorferi sensu lato group with its species B burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, which are transmitted by ticks of the species Ixodes. Laboratory testing of Lyme borreliosis includes culture, antibody detection using ELISA with whole extracts or recombinant chimeric borrelia proteins, immunoblot, and PCR with different levels of sensitivity and specificity for each test. Common skin manifestations of Lyme borreliosis include erythema migrans, lymphocytoma, and acrodermatitis chronica atrophicans. The last two conditions are usually caused by B garinii and B afzelii, respectively, which are seen more frequently in Europe than in America. Late extracutaneous manifestations of Lyme borreliosis are characterised by carditis, neuroborreliosis, and arthritis. We present evidence-based treatment recommendations for Lyme borreliosis and review the prevention of Lyme borreliosis, including the Lyme vaccines.

PMID: 12901891 [PubMed - indexed for MEDLINE]

70. J Med Microbiol. 2003 Jul;52(Pt 7):563-7.

Improved serodiagnosis of erythema migrans using novel recombinant borrelial BBK32 antigens.

Lahdenne P, Panelius J, Saxen H, Heikkilä T, Sillanpää H, Peltomaa M, Arnez M, Huppertz HI, Seppälä IJ.

Hospital for Children and Adolescents, University of Helsinki, Stenbäckinkatu 11, 00290 Helsinki, Finland.

The performances of recombinant borrelial BBK32 proteins as antigens in the serology of erythema migrans (EM) were evaluated in an ELISA. Serum samples were obtained from 75 patients from different geographic areas where three borrelial species, Borrelia burgdorferi sensu stricto, Borrelia afzelii or Borrelia garinii, cause Lyme borreliosis. Antibodies to variant BBK32 proteins were compared with anti-flagella or with anti-IR(6) peptide antibodies. In IgG ELISA at presentation of EM, 65/75 (87 %) patients had antibodies to one or more variants of BBK32, 29/75 (39 %) had antibodies to flagella and 29/75 (39 %) had antibodies to the VlsE IR(6) peptide antigen. The immunoreactivity against variant BBK32 proteins differed in patients from different geographic regions. The present results suggest that the BBK32 proteins used in combination or in parallel may improve the laboratory diagnosis of EM.

PMID: 12808077 [PubMed - indexed for MEDLINE]

71. J Infect Dis. 2003 Jun 15;187(12):1888-94. Epub 2003 Jun 4.

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

Heikkilä T, Huppertz HI, Seppälä I, Sillanpää H, Saxen H, Lahdenne P.

Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland.

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.

PMID: 12792865 [PubMed - indexed for MEDLINE]

72. J Infect Dis. 2003 Apr 15;187(8):1187-99. Epub 2003 Apr 2.

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.

Bacon RM, Biggerstaff BJ, Schriefer ME, Gilmore RD Jr, Philipp MT, Steere AC, Wormser GP, Marques AR, Johnson BJ.

Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA.

Comment in J Infect Dis. 2004 May 15;189(10):1962; author reply 1962-4.

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.

PMID: 12695997 [PubMed - indexed for MEDLINE]

73. Wien Klin Wochenschr. 2002 Jul 31;114(13-14):580-5.

Development and laboratory evaluation of a new recombinant ELISA for the serodiagnosis of Lyme disease.

Hunfeld KP, Ernst M, Zachary P, Jaulhac B, Sonneborn HH, Brade V.

Institute of Medical Microbiology, University Hospital of Frankfurt/Main, Germany.

OBJECTIVE: The use of recombinant proteins for serologic testing represents a modern approach for the improved laboratory diagnosis of Lyme disease (LD). The aim of the present study was to develop and evaluate a new recombinant ELISA (RE) for the detection of specific IgG and IgM antibodies against Borrelia burgdorferi. MATERIALS AND

METHODS: The RE (Biotest AG, Dreieich, Germany) uses mixtures of recombinant p100, OspC, p18 of Borrelia afzelii and a fusion protein of recombinant internal fragments of the flagellum protein (p41) of Borrelia garinii strain PBi and Borrelia afzelii strain PKo. Serologic testing was performed on a commercially available ELISA processor without pre-absorption of sera. The sensitivity of the RE was determined by testing 226 sera obtained from patients suffering from Lyme disease (stage I: n = 148, stage II: n = 35, stage III: n = 43). Specificity of the RE was evaluated in 1107 sera from healthy blood donors and 275 sera from patients with other infectious diseases or autoimmune illnesses (leptospirosis: n = 53, syphilis: n = 70, toxoplasmosis: n = 60, herpes simplex virus: n = 30, HIV: n = 30; rheumatoid-factor positive: n = 32). In addition, 394 routine samples were prospectively tested in comparison with a well established whole-cell lysate extract ELISA (ENZYGNOST Borreliosis, DadeBehring, Germany) for relative sensitivity and specificity.

RESULTS: Overall specificity, determined in 1107 healthy blood donors and 275 sera from patients with other diseases, was 94% for IgG and 91% for IgM. The overall sensitivities in 226 sera obtained from patients suffering from different stages of LD were 67-95%. Moreover, as revealed by prospective testing of 394 routine samples, the relative sensitivity of the RE in comparison with an established whole-cell lysate extract ELISA in the detection of seropositive samples was 81.1% for IgG and 86.5% for IgM with a relative specificity of 98.5% and 93% respectively. The ELISAs showed an overall agreement of 97% for IgG and 92.4% for IgM test results. CONCLUSION: The RE proved to be a reliable and specific screening test in the routine serodiagnosis of LD. In addition, the RE is easy to perform and requires no pre-absorption.

PMID: 12422605 [PubMed - indexed for MEDLINE]

74. J Med Microbiol. 2002 Sep;51(9):731-9.

Recombinant OspC from Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii in the serodiagnosis of Lyme borreliosis.

Panelius J, Lahdenne P, Heikkilä T, Peltomaa M, Oksi J, Seppälä I.

Haartman Institute, Department of Bacteriology and Immunology, Turku University Central Hospital, Finland.

Genes for the outer-surface protein C (OspC) from three north European human isolates of Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii were cloned and sequenced. Polyhistidine-tagged recombinant OspC (rOspC) proteins were produced in Escherichia coli and used, after biotinylation, as antigens on streptavidin-coated plates in enzyme-linked immunosorbent assays (ELISA). In IgM ELISA, 30% (5/17) and 35% (6/17) of patients with erythema migrans (EM) in the acute or convalescent phase, respectively, reacted with one to three rOspCs. Of the patients, 53% (8/15) with neuroborreliosis (NB) and 53% (8/15) with Lyme arthritis (LA) had IgM antibodies to OspC. The immunoreactivity was stronger against rOspC from B. afzelii and B. garinii than against rOspC from B. burgdorferi sensu stricto. In early Lyme borreliosis (LB), rOspC and flagella performed equally well in detecting IgM antibodies. Cross-reactive antibodies to rOspC were observed in serum samples from patients with rheumatoid factor positivity and with syphilis or Epstein-Barr virus (EBV) infection. In IgM ELISA, thiocyanate in the serum dilution buffer reduced EBV-associated non-specific positive reactions. Of the patient sera examined in IgG ELISA, 30% (5/17) with EM in the acute phase, 35% (6/17) with EM in the convalescent phase, 33% (5/15) with NB and 60% (9/15) with LA were positive. Because of the heterogeneity of OspC, a polyvalent antigen with several OspC variants from at least B. afzelii and B. garinii is needed to improve the sensitivity of OspC ELISA in the serodiagnosis of LB in Europe.

PMID: 12358063 [PubMed - indexed for MEDLINE]

75. Pol J Vet Sci. 2002;5(2):71-7.

The use of chosen serological diagnostic methods in Lyme disease in horses. Part I. Indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA).

Dzierzecka M, Kita J.

Department of Infectious Diseases, Faculty of Veterinary Medicine, Warsaw Agricultural University (SGGW), Grochowska 272, 03-849 Warsaw, Poland.

The investigations aimed to establish the reliability of the chosen serological tests designed for the diagnosis of Lyme borreliosis in horses. The investigations were carried out in five Horse Breeding Centres (OHK). Statistical analysis methods were used to determine sample size for particular centres: Krasne (Kr)--49, Łack (Ł)--21, Walewice (W)--111, BogusŁawice (B)--17, Kozienice (K)--61. The experimental material comprised the chosen horses from which blood samples were collected in order to obtain sera. The test used for indirect immunofluorescence assay (IFA No 75941, Bio-Mérieux) is commercially designed for the investigation of human sera and thus needed a prior species adaptation and standardization; ELISA (MRL DIAGNOSTICS, No EL0400G) which was also species adapted and stardandized and ELISA commercially assigned for the examination of dog or horse sera (Die System Diagnostica GmbH Borrelia burgdorferi Veterinary ELISA No. 122.00 Genzyme Virotech GmbH). In the IFA test the highest share of positive results was obtained in respect of the sera from OHK in (K)--60.7% and then in (B)--52.9%, (Ł)--42.9%, (W)--40.5%, (Kr)--38.7%. In the standardized ELISA the highest percent of positive results, amounting to 33.3%, was obtained in respect of the sera from (Ł), and then from (W)--20.7%, (K)--11.5%, (Kr)--10.2% and (B)--5.9%. The percent of positive results obtained in the commercial ELISA also agreement on a high level: the sera originating from (W) were positive in 18.9%, from (K)--9.8%, (Ł)--9.5%. (B)--5.9% and (Kr)--4.1%. Both ELISAs showed high agreement although the standardized test was characterized by a greater tendency for suggesting the presence of B. burgdorferi infection and the agreement of these two ELISAs with the IFA was not so strong. The IFA showed the highest tendency for suggesting the presence of the B. burgdorferi infection, being characterized by the highest percent of false positive results.

PMID: 12189952 [PubMed - indexed for MEDLINE]

76. J Med Microbiol. 2002 Aug;51(8):649-55.

Comparative reactivity of human sera to recombinant VlsE and other Borrelia burgdorferi antigens in class-specific enzyme-linked immunosorbent assays for Lyme borreliosis.

Magnarelli LA, Lawrenz M, Norris SJ, Fikrig E.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504, USA.

A comparative study of human sera was conducted to determine which purified preparations of 11 recombinant antigens of Borrelia burgdorferi sensu stricto were diagnostically most important in enzyme-linked immunosorbent assays (ELISAs). To assess sensitivity, 20 serum samples obtained 1-6 weeks after onset of illness from 20 persons who had physician-diagnosed erythema migrans (EM) were tested for IgM and IgG antibodies. In tests for IgM antibody, seropositivity of > or = 25% was recorded when ELISAs had separate preparations of protein (p) 37, p41-G, outer-surface protein (Osp) C, OspE, OspF or VlsE antigens. Sera reacted most frequently (80% positive) with VlsE antigen in analyses for IgG antibodies. When results of both class-specific assays were considered for VlsE, OspC or OspF, 90% of the EM cases were serologically confirmed. Results of specificity testing with a further 59 sera from persons who had syphilis, louse-borne relapsing fever, oral infections, rheumatoid arthritis or human granulocytic ehrlichiosis and 28 normal sera indicated no false positive reactions when VlsE antigen was used in tests for IgM antibody. One of the 11 louse-borne relapsing fever sera cross-reacted with VlsE antigen in tests for IgG antibodies. Minor cross-reactivity also occurred when p37, OspC, OspE or OspF antigens were used. Overall, VlsE was the most suitable antigen for laboratory diagnosis of Lyme borreliosis during the early weeks of B. burgdorferi infection because of its high sensitivity and specificity.

PMID: 12171295 [PubMed - indexed for MEDLINE]

77. Int J Med Microbiol. 2002 Jun;291 Suppl 33:120-4.

Evidence of a Lyme borreliosis infection from the viewpoint of laboratory medicine.

Lange R, Seyyedi S.

Hospital Dienstleistung + Beratung GmbH, Department of laboratory medicine, Bernau, Germany.

More than 20 years after the first description of the pathogen Borrelia burgdorferi the diagnosis of Lyme borreliosis is based primarily on clinical criteria. Multiple differential diagnoses have to be considered and laboratory confirmation based on various test procedures is required until now. For screening assays the immunofluorescence assay (IFA), enzyme linked immunoassay (EIA), or indirect hemagglutination assay (IHA) are frequently in use. During the last ten years a stepwise diagnostic procedure in USA and Europe has been recommended. The use of a serological screening assay as the first diagnostic step, followed by a confirmatory assay only in the case of a positive or borderline result of the screening assay is now "state of the art". The most frequently used method is a sensitive enzyme linked immunoassay followed by a sensitive and specific immunoblot technique. However, the quality of these indirect serological tests and, mbst importantly, the interpretation criteria of the commercial tests vary dramatically. The choice of Borrelia strains, antigen preparation, production conditions and test procedures vary widely. The immunoblot, used as a confirmatory assay, must meet the highest quality standards but in practice under routine laboratory conditions, these levels are often not reached. The major reason for this "shortcoming" is that Lyme borreliosis is not a sexually transmitted disease and no official approval has been required by the commercial suppliers so far. Another increasingly important aspect is the introduction of molecular biological assays into laboratory routines. Numerous authors published many articles about the usefulness of polymerase chain reaction as a diagnostic tool for the detection of borreliae in human specimens. However, a routine method has not yet been established. Furthermore, no standardized method for DNA isolation from different human specimens has been developed so far. For this reason PCR is not accepted as a routine method and the application should be restricted to a few laboratories with experience in the field of Lyme borreliosis. In summary, it is important to inform clinicians about the limitations of the tests used and it would be very helpful to stage a European or worldwide conference to establish accepted rules for the diagnosis of Lyme borreliosis.

PMID: 12141736 [PubMed - indexed for MEDLINE]

78. Int J Med Microbiol. 2002 Jun;291 Suppl 33:114-9.

Microbiological diagnosis in Lyme borreliosis.

Wilske B.

Max von Pettenkofer Institut für Hygiene und Medizinische Mikrobiologie, LMU München, Germany.

For the microbiological diagnosis of Lyme borreliosis, antibody detection methods are mainly used. Serological diagnosis should follow the principle of a rational stepwise diagnosis (see English Internet Version ( of MiQ Lyme borreliosis). A screening assay (a sensitive ELISA differentiating IgM and IgG) is recommended as the first step. When the ELISA is reactive, it is followed by immunoblot (IgM, IgG) (second step). The reactive diagnostic bands should be clearly identified; this is easy if recombinant antigens are used. Identification of diagnostic bands is difficult in the conventional blot and should be done by monoclonal antibodies. Progress has been made in the sensitivity of the recombinant blot using additional antigens as p58 and homologues of Osp17. In neuroborreliosis the determination of the CSF/serum index is indicated (investigation of paired serum and CSF from the same day). Detection of the etiological agent by culture or PCR should be performed only in specialised laboratories and is confined to specific indications. Recommended specimens are skin, other biopsies, CSF and synovial fluid. The best results are obtained from biopsies (culture and PCR) and synovial fluid (PCR).

PMID: 12141735 [PubMed - indexed for MEDLINE]

79. Clin Diagn Lab Immunol. 2002 Jul;9(4):919-20.

In vitro assessment of antiborrelial activity of OspA vaccine sera.

Fawcett PT, Rose CD, Gibney KM.

Immunology Laboratory, Department of Research, A. I. duPont Hospital for Children, Wilmington, Delaware 19803, USA.

Prevention of Lyme disease by the recombinant OspA-based vaccine reportedly works by preventing transmission of spirochetes from ticks to humans. We report on an in vitro microculture assay, which can be used to provide an indicator of the need for booster doses of vaccine.

PMCID: PMC120022 PMID: 12093696 [PubMed - indexed for MEDLINE]

80. Med Clin North Am. 2002 Mar;86(2):311-40.

Laboratory testing for suspected Lyme disease.

Bunikis J, Barbour AG.

Departments of Medicine and Microbiology and Molecular Genetics, University of California-Irvine, Irvine, California, USA.

Laboratory testing for B. burgdorferi infection is intended to substantiate a physician's clinical judgment of whether a patient has Lyme disease or not. Cultivation of B. burgdorferi from a patient's skin or blood is the gold standard for demonstration of active infection, but it is expensive and lacks clinical sensitivity. Detection of spirochetal DNA in clinical samples by PCR has better sensitivity, but PCR for B. burgdorferi has not yet been standardized for more routine diagnostic testing. Detection of antibodies to B. burgdorferi is the most practical and common approach for laboratory work-up of a case of suspected Lyme disease. Serologic assays fall short of 100% sensitivity and specificity, however, and examination of a single specimen in time does not discriminate between previous and ongoing infection. Because of a background false positivity even among healthy populations of nonendemic regions, serologic testing is recommended only when there is at least a one in five chance, in the physician's estimation, that the patient has active Lyme disease. The pretest likelihood of the disease is determined by the physician in the context of epidemiologic and clinical facts of the case. This estimate can serve to reassure patients who are at low risk of B. burgdorferi infection but are seeking a Lyme test for complaints of a more nonspecific nature. Although new subunit serologic assays based on recombinant proteins are becoming available commercially, the longstanding two-test approach, in which a positive or indeterminate result with a standardized, sensitive ELISA test is followed by verification with a more specific Western blot assay, still provides the physician with a reasonably accurate and reliable assessment of the presence of antibodies to B. burgdorferi. More recent challenges for serologic testing are seropositivity in the population as the result of immunization with the Lyme disease vaccine and the emergence of new Borrelia species that cause Lyme disease-like illnesses.

PMID: 11982304 [PubMed - indexed for MEDLINE]

81. J Clin Microbiol. 2002 Feb;40(2):453-60.

Species-specific serodiagnosis of Lyme arthritis and neuroborreliosis due to Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii by using decorin binding protein A.

Heikkilä T, Seppälä I, Saxen H, Panelius J, Yrjänäinen H, Lahdenne P.

Hospital for Children and Adolescents, University of Helsinki, Finland.

The antigenic potential of decorin binding protein A (DbpA) was evaluated in serodiagnosis of human Lyme borreliosis (LB). The dbpA was cloned and sequenced from the three pathogenic Borrelia species common in Europe. Sequence analysis revealed high interspecies heterogeneity. The identity of the predicted amino acid sequences was 43 to 62% among Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii. The respective recombinant DbpAs (rDbpAs) were produced and tested as antigens by Western blotting and enzyme-linked immunosorbent assay (ELISA). One hundred percent of patients with neuroborreliosis (NB) and 93% of patients with Lyme arthritis (LA) reacted positively. Sera from the majority of patients reacted with one rDbpA only and had no or low cross-reactivity to other two variant proteins. In patients with culture-positive erythema migrans (EM), the sensitivity of rDbpA immunoglobulin G (IgG) or IgM ELISA was low. The DbpA seems to be a sensitive and specific antigen for the serodiagnosis of LA or NB, but not of EM, provided that variants from all three pathogenic borrelial species are included in the combined set of antigens.

PMCID: PMC153353 PMID: 11825956 [PubMed - indexed for MEDLINE]

82. Bratisl Lek Listy. 2001;102(10):454-7.

Significance of specific antibody determination in Lyme borreliosis diagnosis.

Bazovska S, Kondas M, Simkovicova M, Kmety E, Traubner P.

Institute of Epidemiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia.

The diagnosis of Lyme borreliosis, except in cases characterised by pathognomonic clinical manifestation, usually requires confirmation by means of microbiological diagnostic assay, mainly by antibody detection methods. In our study antibodies to B. burgdorferi were tested in neurological patients with suspected Lyme borreliosis, depending on syndrome and clinical diagnosis. Antibodies were tested with IFT, ELISA and immunoblot. Blood samples of patients tested with IFT and ELISA tests were positive in 88 patients. Positive indirect immunofluorescence tests were found in 83 patients; in 5 patients the antibody level was borderline. Of these, 40 were positive also in ELISA but a correlation between IF titers and ELISA-positivity was not established. The immunoblot method confirmed specific antibody positivity in 36 of 88 patients (45.45%) who were positive (or borderline positive) in the indirect IF test, and in 28 of 40 (70%) ELISA-positive patients. Antibody specificity was found in 8 indirect IF-positive patients who were ELISA negative. This may be explained by the higher immunoblot sensitivity in comparison with ELISA. The Lyme borreliosis diagnosis was clinically established in 19 patients; antibodies to B. burgdorferi were only found in 13 patients in all three tests, and in 4 patients only in the indirect IF test. The results of serological tests for antibodies to B. burgdorferi should be interpreted with caution, as the tests are not standardized and may show false positive or false negative results. A two-step serological examination with the immunoblot test is recommended, whereby some nonspecific reactions may be eliminated. The results of serological tests have only supportive value and cannot be deemed conclusive when establishing an etiological diagnosis.

PMID: 11802291 [PubMed - indexed for MEDLINE]

83. J Clin Microbiol. 2002 Jan;40(1):193-7.

Recombinant assay for serodiagnosis of Lyme disease regardless of OspA vaccination status.

Gomes-Solecki MJ, Wormser GP, Schriefer M, Neuman G, Hannafey L, Glass JD, Dattwyler RJ.

Brook Biotechnologies, Inc., Stony Brook, New York 11790-3350, USA.

All current seroassays using cultured Borrelia burgdorferi as their antigen source have been rendered obsolete by the recombinant OspA Lyme disease vaccine. OspA is the major outer surface protein expressed in cultured B. burgdorferi, and any seroassay that uses whole organisms as its antigen source cannot differentiate between subjects who received the vaccine and those who were naturally infected. We developed a new sensitive and specific enzyme-linked immunosorbent assay (ELISA) utilizing recombinant chimeric borrelia proteins devoid of OspA (rNon-OspA) that can be used to detect antibodies to diagnostically important B. burgdorferi antigens in both OspA-vaccinated and nonvaccinated individuals. We tested sera from patients with Lyme disease and with conditions associated with false-positive serologies, OspA-vaccinated individuals, and healthy high-risk workers from an area of endemicity and normal sera from individuals from areas of nonendemicity. The rNon-OspA test was compared with two commercially available whole-cell immunoassays. The rNon-OspA assay is as sensitive and specific as the whole-cell assay (P > 0.05) for detection of anti-B. burgdorferi antibodies. However, the rNon-OspA assay can differentiate between populations comprised of naturally infected and OspA-vaccinated individuals (P < 0.05). Our data demonstrate that this new sensitive rNon-OspA ELISA can be used for the laboratory detection of B. burgdorferi antibodies regardless of vaccination status and could replace existing serologic assays for Lyme disease.

PMCID: PMC120112 PMID: 11773115 [PubMed - indexed for MEDLINE]

84. Epidemiol Mikrobiol Imunol. 2001 Nov;50(4):147-56.

[Criteria for evaluation of immunoblots using Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto for diagnosis of Lyme borreliosis].

[Article in Czech]

Honegr K, Havlasová J, Gebouský P, Dostál V, Pellantová V, Skrabková Z, Hulínská D.

Infekcní klinika FN, Hradec Králové.

The immunoblot was prepared from genotypes Borrelia afzelii (KC 90), Borrelia garinii (M 192) and Borrelia burgdorferi sensu stricto (B 31). Sera of 63 patients with different forms of Lyme borreliosis were examined and 40 healthy donors in the endemic area of the disease. In class IgM in the group of patients significantly more frequently antibodies against OspC, p39, p41 B. afzelii, p39, p41, p66, p83 B. garinii and OspC1, OspA, B. burgdorferi sensu stricto were found. In class IgG there were antibodies against p39, p41, p93 B. afzelii, p14, p41, p93 B. garinii and OspA, OspC p93 B. burgdorferi sensu lato. Based on the assembled results by means of discrimination analysis and logistic regression the most suitable combinations of antigens for evaluation of immunoblots in different genotypes were determined. Furthermore evaluation was suggested using a combination of antigens of several genotypes which led to an increased sensitivity and specificity of the immunoblot. Tables were prepared for easier evaluation of newly examined sera samples.

PMID: 11769176 [PubMed - indexed for MEDLINE]

85. J Clin Microbiol. 2001 Nov;39(11):4013-9.

Recombinant flagellin A proteins from Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii in serodiagnosis of Lyme borreliosis.

Panelius J, Lahdenne P, Saxen H, Heikkilä T, Seppälä I.

Haartman Institute, Department of Bacteriology and Immunology, Helsinki, Finland.

Genes for flagellin A (FlaA) proteins from European borrelial strains of Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii were cloned and sequenced. An identity of 92 to 93% was observed in the flaA sequences of the different species. Polyhistidine-tagged recombinant FlaA (rFlaA) proteins were produced in Escherichia coli and used as antigens in Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). In immunoglobulin G (IgG) WB, 71% (10 of 14) of the sera from neuroborreliosis and 86% (12 of 14) of those from Lyme arthritis patients reacted with one to three rFlaAs. In IgG ELISA, 74% (14 of 19) and 79% (15 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. The immunoreactivity in local European patient sera was stronger against rFlaA from B. garinii and B. afzelii than against rFlaA from B. burgdorferi sensu stricto. Neither IgG nor IgM ELISA was sensitive in the serodiagnosis of erythema migrans. Serum samples from patients with syphilis and systemic lupus erythematosus showed mild cross-reactivity in IgG tests. Sera from Yersinia enterocolitica or beta-hemolytic Streptococcus infections showed only occasional responses. With IgM ELISA, 58% (11 of 19) and 37% (7 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. Cross-reactive antibodies to FlaA, especially in serum samples from patients with rheumatoid factor positivity and Epstein-Barr virus infection, reduced the specificity of IgM serodiagnosis. Therefore, rFlaA seems to have a limited role for IgM serodiagnosis, yet rFlaA might be useful in the IgG serodiagnosis of disseminated Lyme borreliosis.

PMCID: PMC88480 PMID: 11682523 [PubMed - indexed for MEDLINE]

86. Arch Intern Med. 2001 Sep 10;161(16):2015-20.

A first-tier rapid assay for the serodiagnosis of Borrelia burgdorferi infection.

Gomes-Solecki MJ, Wormser GP, Persing DH, Berger BW, Glass JD, Yang X, Dattwyler RJ.

Brook Biotechnologies, Inc, Stony Brook, NY, USA.

BACKGROUND: The present recommendation for the serologic diagnosis of Lyme disease is a 2-tier process in which a serum sample with a positive or equivocal result by an enzyme-linked immunosorbent assay (ELISA) or immunofluorescent assay is then followed by supplemental testing by Western blot. Our laboratory has developed recombinant chimeric proteins composed of key Borrelia epitopes. These novel antigens are consistent and are easily standardized.

METHODS: We adapted these recombinant proteins into a new immunochromatographic format that can be used as a highly sensitive and specific first-tier assay that can be used to replace the ELISA or immunofluorescent assay.

RESULTS: This rapid test was equally sensitive (P>.05) and more specific (P<.05) than a frequently used commercial whole cell ELISA. The overall clinical accuracy achieved on agreement studies among 3 Lyme research laboratories on clinically defined serum panels was shown to be statistically equivalent to the commercial ELISA. The assay can detect anti-Borrelia burgdorferi antibodies in either serum or whole blood. CONCLUSION: This sensitive and specific rapid assay, which is suited for the physician's office, streamlines the 2-tier system by allowing the physician to determine if a Western blot is necessary at the time of the initial office visit.

PMID: 11525704 [PubMed - indexed for MEDLINE]

87. J Clin Microbiol. 2001 May;39(5):2039-40.

Recombinant low-molecular-mass proteins pG and LA7 from Borrelia burgdorferi reveal low diagnostic sensitivity in an enzyme-linked immunosorbent assay.

Rauer S, Wallich R, Neubert U.

PMCID: PMC88083 PMID: 11388171 [PubMed - indexed for MEDLINE]

88. Minerva Med. 2001 Feb;92(1):29-33.

[Reliability of a polymerase chain reaction (PCR) technique in the diagnosis of Lyme borreliosis].

[Article in Italian]

Grignolo MC, Buffrini L, Monteforte P, Rovetta G.

DISEM, Cattedra di Reumatologia, Istituto E. Bruzzone, ASL n. 3 Genovese, Centro Reumatologico, Gruppo Italiano per lo Studio della Malattia di Lyme (GISML), Università degli Studi, Genoa, Italy.

BACKGROUND: The Polymerase Chain Reaction (PCR) technique has some application limits therefore suggesting the evaluation of the sensibility (SE), specificity (SP), predictive positive value (PPV) and predictive negative value (NPV) of a PCR test detecting a target sequence of OspA gene of Borrelia burgdorferi sensu lato. This technique is currently used in the diagnosis of Lyme borreliosis in our laboratory.

METHODS: Ninety-three patients, either examined in our outpatient clinic or coming from other clinical centres, were divided in two groups on the basis of a correlation between clinical picture and serologic tests (ELISA and Western Blot): 42 patients were true positives (tp) and 51 true negatives (tn). The samples (77 sera, 16 cerebrospinal fluids) have been tested with the above PCR technique. The results obtained were used to calculate the SE, SP, PPV and NPV. Subsequently, 13 urine samples of patients with negative serum PCR were examined with the same technique.

RESULTS: A high SP and a low SE, high PPV and a considerably lower NPV have been reported. 50% of the PCR positive results, obtained with serum and cerebrospinal fluid samples corresponded to patients who were true positives at clinical examination but negatives at serologic tests. 62.5% of urine samples positive results belonged to tp patients who had negative serologic and serum PCR RESULTS.

CONCLUSIONS: The obtained results suggested a good reliability of positive results obtained with the PCR technique used in this study and allowed the false negatives of serologic tests to be detected, more specifically when urine samples were used.

PMID: 11317136 [PubMed - indexed for MEDLINE]

89. Br J Ophthalmol. 2001 May;85(5):552-5.

Detection of Borrelia burgdorferi DNA in urine of patients with ocular Lyme borreliosis.

Pleyer U, Priem S, Bergmann L, Burmester G, Hartmann C, Krause A.

Department of Ophthalmology, Charité, Humboldt University, Campus Virchow Hospital, Augustenburger Platz 1, D-13353 Berlin, Germany.

AIM: To evaluate the diagnostic value of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi DNA in patients with ocular Lyme borreliosis.

METHODS: Of 256 consecutive uveitis patients six selected individuals with clinical evidence for Lyme borreliosis and 30 patients with non-Lyme uveitis were enrolled. Lyme serology was performed by ELISA and western blotting. Urine samples were examined by an optimised nested polymerase chain reaction (PCR) protocol.

RESULTS: Only four of six uveitis patients suspected for Lyme borreliosis were ELISA positive, while all six subjects showed a positive western blot. B burgdorferi PCR was positive in all of these six patients. Whereas two of the 30 controls had a positive Lyme serology, B burgdorferi DNA was not detectable by PCR in any sample from these patients.

CONCLUSIONS: PCR for the detection of B burgdorferi DNA in urine of uveitis patients is a valuable tool to support the diagnosis of ocular Lyme borreliosis. Moreover, these patients often show a weak humoral immune response which may more sensitively be detected by immunoblotting.

PMCID: PMC1723951 PMID: 11316715 [PubMed - indexed for MEDLINE]

90. Scand J Infect Dis. 2001;33(2):128-31.

Borrelia burgdorferi antibodies in outdoor and indoor workers in south-west Sweden.

Werner M, Nordin P, Arnholm B, Elgefors B, Krantz I.

Department of Infectious Diseases, Bords Hospital, Sweden.

Two hundred and fifty-three farmers and forest workers and 249 clerks from south-west Sweden were recruited to a cross-sectional seroprevalence study to find out if individuals working outdoors are more prone to acquire Borrelia burgdorferi infection than indoor workers and to find undiagnosed cases of Lyme borreliosis. The participants answered a questionnaire and blood specimens were collected to estimate the prevalence of antibodies to B. burgdorferi in each group. Sera were analysed with an enzyme-linked immunoassay technique to determine IgG antibodies to B. burgdorferi flagellum. The prevalence of B. burgdorferi antibodies was 7.6% in the farmers and forest workers vs. 5.3% in the clerks (adjusted odds ratio [age, sex] = 1.2 [95% confidence interval = 0.5-2.8]). One case of Lyme borreliosis was diagnosed. The positive predictive value of the antibody test was estimated to be 3% in the studied populations. B. burgdorferi infection is of low endemicity in south-west Sweden and is probably not an occupational risk among outdoor workers. Undiagnosed cases of Lyme borreliosis are uncommon. The test used is not acceptable for screening purposes.

PMID: 11233848 [PubMed - indexed for MEDLINE]

91. Clin Lab. 2001;47(1-2):41-9.

Serodiagnosis of Lyme borreliosis using detection of different immunoglobulin (sub)classes by enzyme-linked immunosorbent assay and Western blotting.

Goossens HA, van den Bogaard AE, Nohlmans MK.

Department of Medical Microbiology, University of Maastricht, The Netherlands.

To improve the performance of enzyme-linked immunosorbent assays for the serodiagnosis of Lyme borreliosis, the prevalence of several immunoglobulin classes and subclasses against various antigens of Borrelia burgdorferi was investigated by Western blotting. The sera of 40 early Lyme borreliosis patients (ELB), 27 late Lyme borreliosis patients (LLB), 62 healthy controls and 140 non-Lyme borreliosis patients were used. Detection of IgG1 versus total IgG was found to be more sensitive in detecting Borrelia burgdorferi antigens, especially flagellin (41 kD) protein, but did not improve the performance of Western blotting. The use of IgG1 detection showed an increase in sensitivity and specificity for the early Lyme borreliosis patient group compared to the standard IgG and IgM detection method by enzyme immunoassays using purified Borrelia burgdorferi flagellum. However, in an enzyme immunoassay using a total sonicate, sensitivity in detecting early Lyme borreliosis and late Lyme borreliosis with IgG1 remained lower compared to the detection of early Lyme borreliosis by IgM antibodies and late Lyme borreliosis by total IgG antibodies.

PMID: 11214222 [PubMed - indexed for MEDLINE]

92. Biopolymers. 2000;55(4):319-33.

Selection of continuous epitope sequences and their incorporation into poly(ethylene glycol)-peptide conjugates for use in serodiagnostic immunoassays: application to Lyme disease.

Qiu B, Brunner M, Zhang G, Sigal L, Stein S.

Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, USA.

Continuous epitope sequences were selected from immunogenic Bb proteins by epitope mapping. The identified epitope sequences were synthesized by solid phase peptide synthesis and purified by high performance liquid chromatography. Each epitope was conjugated individually to a multifunctional poly(ethylene glycol) (PEG) carrier. The result PEG-peptide conjugates were used as antigens in ELISA for diagnosis of Lyme disease. The results showed that the defined epitope peptides were Lyme disease specific and could be used in a format of PEG-peptide conjugate as the antigen to achieve improved sensitivity and specificity.

PMID: 11169923 [PubMed - indexed for MEDLINE]

93. Z Kardiol. 2000 Nov;89(11):1046-52.

[Acute myocarditis and cardiomyopathy in Lyme borreliosis].

[Article in German]

Scheffold N, Sucker C, Bergler-Klein J, Kaag N, Cyran J.

Medizinische Klinik I Schwerpunkt Kardiologie Klinikum Heilbronn Akademisches Lehrkrankenhaus der Universität Heidelberg Am Gesundbrunnen 20-24 D-74078 Heilbronn.

Heart involvement of Lyme disease occurs in about 4-10% of patients with Lyme borreliosis. The most common manifestation is acute, self-limiting Lyme carditis, which manifests mostly as transient conduction disorders of the heart, pericarditis and myocarditis. Laboratory tests (ELISA, immunoblotting and PCR) usually have limited sensitivity and specificity, and criteria of performance and interpretation have not yet been fully evaluated. Therefore the laboratory evidence should only be interpreted in conjunction with other clinical and diagnostic features. Recently there has been convincing evidence published that long standing dilated cardiomyopathy in many cases is associated with a chronic Borrelia burgdorferi (BB) infection. Several studies showed a higher prevalence of BB antibodies in patients with severe heart failure in endemic areas (e.g., 26% versus 8% in healthy individuals). The isolation of spirochetes from the myocardium gave further evidence that BB may cause chronic heart muscle disease. In several studies antimicrobial treatment showed an improvement of the left ventricular function in patients with dilated cardiomyopathy associated with BB. However the duration of dilated cardiomyopathy before treatment plays an important part in the clinical outcome of BB-associated chronic myocarditis.

PMID: 11149272 [PubMed - indexed for MEDLINE]

94. Clin Diagn Lab Immunol. 2001 Jan;8(1):150-60.

Identifying diagnostic peptides for lyme disease through epitope discovery.

Kouzmitcheva GA, Petrenko VA, Smith GP.

Division of Biological Sciences, Tucker Hall, University of Missouri, Columbia, Missouri 65211, USA.

Serum antibodies from patients with Lyme disease (LD) were used to affinity select peptide epitopes from 12 large random peptide libraries in phage display format. The selected peptides were surveyed for reactivity with a panel of positive sera (from LD patients) and negative sera (from subjects without LD), thus identifying 17 peptides with a diagnostically useful binding pattern: reactivity with at least three positive sera and no reactivity with any of the negative sera. The peptides define eight sequence motifs, none of which can be matched convincingly with segments of proteins from Borrelia burgdorferi, the LD pathogen; evidently, then, they are "mimotopes," mimicking natural pathogen epitopes without matching contiguous amino acids of pathogen proteins. Peptides like these could be the basis of a new diagnostic enzyme-linked immunosorbent assay for LD, with sufficient specificity and sensitivity to replace expensive immunoblotting tests that are currently required for definitive serological diagnosis. Moreover, the method used to discover these peptides did not require any knowledge of the pathogen and involved generic procedures that are applicable to almost any infectious disease, including emerging diseases for which no pathogen has yet been identified.

PMCID: PMC96025 PMID: 11139210 [PubMed - indexed for MEDLINE]

95. J Med Microbiol. 2000 Oct;49(10):911-5.

False-negative serology in patients with neuroborreliosis and the value of employing of different borrelial strains in serological assays.

Kaiser R.

Neurologische Klinik und Poliklinik der Albert-Ludwigs-Universität Freiburg, Germany.

The risk of obtaining false-negative results in serological assays in serum and CSF specimens with only one strain of Borrelia burgdorferi sensu lato as antigen was investigated in 79 patients with neuroborreliosis with specimens obtained at initial presentation. Serum antibodies were assessed by immunoblotting; the criteria of Hauser et al. were used to evaluate the test. The intrathecal synthesis of borrelial-specific IgM and IgG antibodies was examined by enzyme immunoassay (EIA). Strains of B. burgdorferi sensu stricto (BbZ160), B. garinii (Bbii50) and B. afzelii (PKO) served as sources of antigen in both assays. All patients produced either a positive IgM or IgG test in serum with at least one strain of B. burgdorferi sensu lato. Reactivity of IgM or IgG antibodies, or both, with antigens of all three strains was demonstrated in 67 (85%) of 79 sera. The correlation of results of immunoblotting with different strains was significantly better for IgG (85%) than for IgM antibodies (54%). The variability of positive IgM reactions in 18 specimens was mainly due to the fact that the antibodies were directed to the relevant variable outer-surface protein C (p23). Intrathecal synthesis of IgG antibodies was demonstrated in 58 patients (81%) of 72 and of IgM antibodies in 25 of 58 patients. No patient had isolated intrathecal synthesis of IgM antibodies. The majority of CSF samples (56 of 58) were assessed as IgG antibody-positive, independent of the borrelial strain used as antigen in EIA, whereas only 10 of 25 IgM antibody-positive CSF specimens reacted with all three strains. All patients in the study had intrathecal antibody synthesis demonstrable at 6-week follow-up. From this study it is concluded that there is a small, but real, risk of false-negative serological findings at the time of initial clinical presentation in patients with typical symptoms of neuroborreliosis. In these patients a negative serological result with one strain should prompt the repetition of the test with other strains of B. burgdorferi sensu lato.

PMID: 11023188 [PubMed - indexed for MEDLINE]

96. FEMS Immunol Med Microbiol. 2000 Sep;29(1):15-21.

Specific immune response to a synthetic peptide derived from outer surface protein C of Borrelia burgdorferi predicts protective borreliacidal antibodies.

Ikushima M, Matsui K, Yamada F, Kawahashi S, Nishikawa SK.

Division of Clinical Microbiology, Saitama Institute of Public Health, Japan.

In a previous study, we described the development of a new specific serodiagnostic test for Lyme disease involving enzyme-linked immunosorbent assay and a synthetic peptide, OspC-I. The OspC-I peptide is derived from part of the outer surface protein C (OspC) amino acid sequence of Borrelia burgdorferi and is located in the region conserved among B. burgdorferi sensu stricto or sensu lato isolates. In this study, we demonstrate that sera containing antibodies against OspC-I from patients with early Lyme disease had borreliacidal activity against isolates of three genospecies of Lyme disease spirochete, B. burgdoreferi B31, B. garinii HPI and B. afzelii HT61. However, the borreliacidal activity against B. burgdorferi, which has not been isolated in Japan, was weaker than that against the other species. Vaccination of mice with OspC-I induced the production of anti-OspC-I antibodies in serum with borreliacidal activity. The immune mouse serum had significantly higher levels of borreliacidal activity against HP1 and HT61, than against B31. Neutralization of borreliacidal activity with anti-IgM antibodies showed that the borreliacidal activity of anti-OspC-I antibodies in serum was due to IgM. Furthermore. mice vaccinated with OspC-I were protected against challenge with HPI and HT61. but not fully protected against infection with B31. These results suggest that OspC-I is not only a specific antigen for use in serodiagnostic tests for Lyme disease, but is also a potential candidate for a Lyme disease vaccine in Japan.

PMID: 10967255 [PubMed - indexed for MEDLINE]

97. J Clin Microbiol. 2000 Jul;38(7):2530-5.

Recombinant chimeric Borrelia proteins for diagnosis of Lyme disease.

Gomes-Solecki MJ, Dunn JJ, Luft BJ, Castillo J, Dykhuizen DE, Yang X, Glass JD, Dattwyler RJ.

Brook Biotechnologies, Inc., Long Island High Technology Incubator, Stony Brook, New York 11790, USA.

Current serologic Lyme disease tests use whole borrelia cells as the source of antigen. These assays are difficult to standardize and to optimize for sensitivity and specificity. To help solve these problems, we constructed a library of recombinant chimeric proteins composed of portions of key antigens of Borrelia burgdorferi. These proteins were then used to develop an enzyme-linked immunosorbent assay. We compared our assay with the most sensitive of three whole-cell borrelia assays. We found that the recombinant assay could detect antibodies significantly better from early Lyme disease sera (P<0.05), and had the same sensitivity for late Lyme disease sera, as the most sensitive whole-cell borrelia assay. On potentially cross-reactive sera, the recombinant assay was more specific, but not significantly so, than the best whole-cell borrelia assay. Optimization of the recombinant assay offers the potential for a significant improvement in both sensitivity and specificity.

PMCID: PMC86960 PMID: 10878038 [PubMed - indexed for MEDLINE]

98. J Clin Microbiol. 2000 May;38(5):1735-9.

Serologic diagnosis of Lyme borreliosis by using enzyme-linked immunosorbent assays with recombinant antigens.

Magnarelli LA, Ijdo JW, Padula SJ, Flavell RA, Fikrig E.

Department of Entomology, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504, USA.

Class-specific enzyme-linked immunosorbent assays (ELISAs) with purified recombinant antigens of Borrelia burgdorferi sensu stricto and Western blot analyses with whole cells of this spirochete were used to test human sera to determine which antigens were diagnostically important. In analyses for immunoglobulin M (IgM) antibodies, 14 (82%) of 17 serum samples from persons who had erythema migrans reacted positively by an ELISA with one or more recombinant antigens. There was frequent antibody reactivity to protein 41-G (p41-G), outer surface protein C (OspC), and OspF antigens. In an ELISA for IgG antibodies, 13 (87%) of 15 serum samples had antibodies to recombinant antigens; reactivity to p22, p39, p41-G, OspC, and OspF antigens was frequent. By both ELISAs, serum specimens positive for OspB, OspE, and p37 were uncommon. Analyses of sera obtained from persons who were suspected of having human granulocytic ehrlichiosis (HGE) but who lacked antibodies to ehrlichiae revealed IgM antibodies to all recombinant antigens of B. burgdorferi except OspB and IgG antibodies to all antigens except OspE. Immunoblotting of sera from the study group of individuals suspected of having HGE reaffirmed antibody reactivity to multiple antigens of B. burgdorferi. There was minor cross-reactivity when sera from healthy subjects or persons who had syphilis, oral infections, or rheumatoid arthritis were tested by ELISAs with p37, p41-G, OspB, OspC, OspE, and OspF antigens. Although the results of class-specific ELISAs with recombinant antigens were comparable to those recorded for assays with whole-cell antigen and for individuals with confirmed clinical diagnoses of Lyme borreliosis, immunoblotting is still advised as an adjunct procedure, particularly when there are low antibody titers by an ELISA.

PMCID: PMC86574 PMID: 10790090 [PubMed - indexed for MEDLINE]

99. J Microbiol Methods. 2000 Mar;40(1):79-88.

Development and evaluation of a broad-range PCR-ELISA assay with Borrelia burgdorferi and Streptococcus pneumoniae as model organisms for reactive arthritis and bacterial meningitis.

Fischer-Romero C, Lüthy-Hottenstein J, Altwegg M.

Department of Medical Microbiology, University of Zürich, Switzerland.

We have developed an assay based on a 16S rDNA broad-range amplification system followed by species-specific detection with a commercially available PCR-ELISA kit. B. burgdorferi and S. pneumoniae were used as model systems for arthritis and meningitis, respectively. The sensitivity of the B. burgdorferi assay was comparable to that of a species-specific PCR, whereas for S. pneumoniae the detection limit was one to three organisms as determined by plate counts. To specifically differentiate two species, two discontinuously located nucleotide differences in the region complementary to the capture probe are required during the detection step with the PCR-ELISA kit. A preliminary clinical evaluation was performed with eight specimens (joint and cerebrospinal fluids) previously shown to contain B. burgdorferi DNA. Except for one sample which was positive by the broad-range PCR-ELISA system only, the results were in agreement with those obtained by B. burgdorferi species-specific PCR. None of the 23 control samples were positive by either method. Thus, broad-range amplification in combination with the PCR-ELISA kit promises to be a sensitive and specific format for the detection of agents causing reactive arthritis, meningitis or other diseases associated with a limited number of different bacteria.

PMID: 10739346 [PubMed - indexed for MEDLINE]

100. J Vet Diagn Invest. 2000 Mar;12(2):126-9.

Canines as sentinels for Lyme disease in San Diego County, California.

Olson PE, Kallen AJ, Bjorneby JM, Creek JG.

Epidemiology Department, Naval Station, San Diego, CA 92136, USA.

Prevalence of Lyme borreliosis in canine sentinels has been shown to correlate with infection in humans. One thousand canine sera (917 dogs, 83 coyotes) obtained from animal control authorities and area veterinarians were screened by ELISA for antibodies to Borrelia burgdorferi. Results were validated by Western blot and indirect fluorescent antibody (IFA) tests at referee laboratories. Criterion for a positive Western blot was presence of 5 of 10 of the most common antigen IgG bands; for IFA, >1:128 or the equivalent when correcting for interlaboratory variability. Twenty-two of 1,000 canines were confirmed serologically positive (21 dogs and 1 coyote; seroprevalence 2.3% and 1.2%, respectively). Lifestyle, breed size, gender, and age were not statistically predictive of seropositive status. No regional clustering of seropositive animals was detected. The low prevalence of seropositivity in sentinel canines suggests the Lyme borreliosis hazard in San Diego County is minimal.

PMID: 10730940 [PubMed - indexed for MEDLINE]

101. Clin Infect Dis. 2000 Mar;30(3):545-8.

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

Wormser GP, Carbonaro C, Miller S, Nowakowski J, Nadelman RB, Sivak S, Aguero-Rosenfeld ME.

Division of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, NY 10595, USA.

Comment in Clin Infect Dis. 2001 Apr 1;32(7):1114.

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

PMID: 10722442 [PubMed - indexed for MEDLINE]

102. J Microbiol Methods. 2000 Apr;40(2):163-73.

An indirect hemagglutination antibody test to detect antibodies to Borrelia burgdorferi in patients with Lyme disease.

Pavia CS, Wormser GP, Bittker S, Cooper D.

NYCOM Microbiology Laboratory of NYIT, Old Westbury, NY 11568, USA.

An indirect hemagglutination antibody (IHA) test was evaluated for its ability to detect borrelial antibodies in serum samples from patients with Lyme disease. The key test reagent developed for this antibody detection system was tannic acid-treated and glutaraldehyde-fixed sheep red blood cells (SRBC) containing Borrelia burgdorferi (Bb) antigens attached to the outer surface of the SRBC. In order to establish suitable cut-off titers, initial specificity and sensitivity measurements were made using sera from 100 anonymous healthy volunteers and 30 additional pre-determined samples known to be non-reactive or reactive for Lyme disease or syphilis. These results were compared with those obtained using a commercially available ELISA. At titers >/=64, the IHA test had a combined 98% specificity and 100% sensitivity for these 130 serum samples, 30 of which were known positives or negatives, whereas the ELISA was less specific (93%) and much less sensitive (80%). Subsequent testing was performed on sera from 65 patients with the erythema migrans (EM) rash and 20 patients with early disseminated (cardiac/neurologic) symptoms or with Lyme arthritis. At initial presentation, 46-48% of the EM patients had IHA reactivity, with titers >/=128, while 42% were positive in the ELISA. Follow-up testing performed on these EM patients, 8-12 days after receiving antibiotic treatment, revealed that Bb antibodies were detected best by the IHA test (83-86% reactive) relative to the ELISA (81% reactive). Bb antibodies were readily detectable on all of the serum samples from the early disseminated and late stage Lyme disease cases in both assay systems. Based on these results and because of its technical and interpretive simplicity, the IHA test should be considered as a useful and convenient alternative for the serological analysis of Bb infections.

PMID: 10699672 [PubMed - indexed for MEDLINE]

103. Wien Klin Wochenschr. 1999 Dec 10;111(22-23):957-60.

Is IgM of diagnostic value in case of delayed intrathecal production of IgG antibodies?

Pierer K, Stünzner D, Feichtinger M, Homann CN, Kleinert G, Kessler HH, Marth E.

Institute of Hygiene, Medical School, Karl Franzens University, Graz, Austria.

The neurological manifestations of Lyme borreliosis comprise a wide range of clinical signs. However, these symptoms might have other aetiologies. Therefore detection of intrathecal production of specific antibodies is necessary to confirm the clinical assumption of neuroborreliosis (NB). In case of delayed intrathecal production of specific IgG antibodies, detection of IgM could play a role in the early diagnosis of NB. To clarify whether IgM is of diagnostic value in such cases, paired CSF serum samples from 176 patients with suspected NB admitted to the department of Neurology, Karl Franzens University, Graz, Austria, were tested. Testing was performed with the IDEA Neuroborreliosis Kit (Dako, Denmark) and Enzygnost Borreliosis (Behring, Germany) and results of both methods were compared. According to well defined criteria 63 of the 176 patients had defined NB and 113 were regarded as possible NB. Twelve out of 63 patients with defined NB had delayed intrathecal IgG production. Only one patient with delayed IgG production had an intrathecal IgM production prior to IgG. In all patients with possible NB no intrathecal production of IgM was detected. At the time of the first lumbar puncture IgG intrathecal production could be detected with the IDEA seven times more often than with the Enzygnost Borreliosis. The determination of intrathecal production of IgM does not appear to be of diagnostic value in patients with delayed IgG antibody production. Therefore a consecutive lumbar puncture is more likely to confirm clinical assumption if there is strong clinical evidence of NB.

PMID: 10666808 [PubMed - indexed for MEDLINE]

104. J Clin Lab Anal. 2000;14(1):20-6.

Antibodies to Borrelia burgdorferi in European populations.

Gutiérrez J, Guerrero M, Nuñez F, Soto MJ, Piédrola G, Maroto MC.

Microbiology Department, Granada University, Granada, Spain.

We compared the antibodies to B. burgdorferi in three different populations in order to evaluate the diagnostic reliability of Lyme borreliosis serologic analysis. The subjects included 25 patients with Lyme borreliosis (Group 1); 50 patients with diseases of unknown cause, B. burgdorferi ELISA-positive in serum and without B. burgdorferi infection (Group 2); and 1,251 individuals without Lyme borreliosis (Group 3). All samples were tested for B. burgdorferi B31 and B. afzelii antigens using ELISA. The positive results of the ELISA B. burgdorferi B31 assay were confirmed with Western blot for the same strain. In Group 3, 162 (12.9%) patients were ELISA positive for B. burgdorferi B31, while only 6 (0.6%) patients had IgG ELISA antibodies to B afzelii. Bands in WB were detected in 104 (8.4%) of the Group 3 subjects. The bands found to be most reliable for the identification of strain B. burgdorferi B31 by IgG WB were those representing the 93, 39, 34, and 23-kDa proteins. Our results show that serologic diagnosis of Lyme borreliosis is far from clearly established. To date, the only reliable criteria are clinical ones correlated with laboratory evidence.

Copyright 2000 Wiley-Liss, Inc.

PMID: 10645981 [PubMed - indexed for MEDLINE]

105. J Clin Microbiol. 2000 Jan;38(1):313-7.

Evaluation of whole-cell and OspC enzyme-linked immunosorbent assays for discrimination of early lyme borreliosis from OspA vaccination.

Wieneke CA, Lovrich SD, Callister SM, Jobe DA, Marks JA, Schell RF.

Microbiology Research Laboratory, Gundersen Lutheran Medical Center, La Crosse, Wisconsin 54601, USA.

A recombinant Lyme borreliosis vaccine consisting of outer surface protein A (OspA) is commercially available for vaccination of humans against infection with Borrelia burgdorferi. Vaccination with OspA induces an antibody response that makes serologic interpretation of infection with B. burgdorferi difficult, especially by screening tests based on whole-cell preparations of B. burgdorferi. We show that an enzyme-linked immunosorbent assay with B. burgdorferi sensu stricto 50772, which lacks the plasmid encoding OspA and OspB, or a full-length recombinant OspC protein can identify patients infected with B. burgdorferi. We found that 69 and 65% of serum samples from patients with case-defined early Lyme borreliosis had anti-B. burgdorferi sensu stricto 50772 and anti-OspC reactivities, respectively. In addition, little or no reactivity was detected with sera obtained from individuals vaccinated with OspA. Unfortunately, 51 and 33% of sera from healthy patients and sera from patients with other illnesses were also reactive against B. burgdorferi sensu stricto 50772 and OspC, respectively. Although these assays can discriminate B. burgdorferi infection from vaccination with OspA, their lack of specificity highlights the necessity for confirming equivocal or positive reactivities with more specific serodiagnostic tests.

PMCID: PMC88715 PMID: 10618107 [PubMed - indexed for MEDLINE]

106. J Clin Microbiol. 1999 Dec;37(12):3997-4004.

Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi.

Lawrenz MB, Hardham JM, Owens RT, Nowakowski J, Steere AC, Wormser GP, Norris SJ.

Departments of Pathology and Laboratory Medicine and Microbiology and Molecular Genetics, University of Texas Medical School at Houston, Houston, Texas 77030, USA.

VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.

PMCID: PMC85865 PMID: 10565921 [PubMed - indexed for MEDLINE]

107. J Clin Microbiol. 1999 Dec;37(12):3990-6.

Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi vlsE.

Liang FT, Steere AC, Marques AR, Johnson BJ, Miller JN, Philipp MT.

Department of Parasitology, Tulane Regional Primate Research Center, Tulane University Medical Center, Covington, Louisiana 70433, USA.

VlsE, the variable surface antigen of Borrelia burgdorferi, contains an immunodominant conserved region named IR(6). In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C(6)) with the IR(6) sequence was explored. Sensitivity was assessed with serum samples (n = 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C(6) peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.

PMCID: PMC85863 PMID: 10565920 [PubMed - indexed for MEDLINE]

108. Rheumatology (Oxford). 1999 Nov;38(11):1121-6.

Clinical evaluation of guidelines and two-test approach for lyme disease.

Blaauw AA, van Loon AM, Schellekens JF, Bijlsma JW.

Department of Rheumatology, University Medical Centre, 3508 GA Utrecht, The Netherlands.

OBJECTIVE: The diagnosis of Lyme disease should be based on objective clinical signs and symptoms. In a clinical study, we have evaluated whether the recommended two-step approach for serodiagnosis of Lyme disease is useful in daily clinical practice and can influence clinical decision making.

METHODS: The signs and symptoms of patients with ongoing musculoskeletal complaints, assumed by their referring physician or themselves to be attributable to active or chronic Lyme disease, and of patients diagnosed as having Lyme disease, were evaluated. On the basis of clinical evaluation only, patients were classified into three groups: previous Lyme disease, active Lyme disease and no Lyme disease. Antibodies to Borrelia burgdorferi were determined by means of an enzyme-linked immunosorbent assay (ELISA), followed, when positive, by immunoblotting.

RESULTS: One hundred and three patients (41 males and 62 females, mean age 48.7 yr) participated in the study. Of the 49 patients classified as previous Lyme disease, 25 (51%) had antibodies to B. burgdorferi. All 10 patients with active Lyme disease had positive antibodies and 12 of the 44 patients (27%) classified as no Lyme disease had positive antibodies. No statistically significant differences were found between the percentage of positive immunoblots from patients with previous Lyme disease (72%) and patients with active Lyme disease (100%). In the group of no Lyme disease, five out of 12 patients had a negative immunoblot. Concerning serological testing, immunoblotting could have added additional information. However, immunoblotting did not influence clinical decision making in this group of patients. CONCLUSION: Immunoblotting did not influence clinical decision making for the 47 patients with antibodies to B. burgdorferi in this study.

PMID: 10556266 [PubMed - indexed for MEDLINE]

109. JAMA. 1999 Jul 7;282(1):62-6.

Role of serology in the diagnosis of Lyme disease.

Brown SL, Hansen SL, Langone JJ.

Division of Postmarket Surveillance, Food and Drug Administration, Center for Devices and Radiological Health, Rockville, MD, USA.

Comment in JAMA. 1999 Jul 7;282(1):79-80.

Numerous concerns regarding the potential for misdiagnosis of Lyme disease using commercial assays have been voiced by the US Food and Drug Administration (FDA). We attempted to clarify the clinical value of serologic testing for Lyme disease using the results of commonly marketed assays for detecting antibody to Borrelia burgdorferi, the organism that causes Lyme disease. We reviewed published studies on B burgdorferi test performance published through 1998, package insert labeling from FDA-cleared test kits for B burgdorferi, and Lyme Disease Survey Set LY-A from the College of American Pathologists. We assessed the sensitivity and specificity of commercial serologic tests (enzyme-linked immunosorbent assay [ELISA], immunofluorescence antibody [IFA], and immunodot) for diagnosis of Lyme disease. To reduce this risk of misdiagnosis, it is important that clinicians understand the performance characteristics and limitations of these tests. These tests, in common use in clinical or commercial laboratories, should be used only to support a clinical diagnosis of Lyme disease, not as the primary basis for making diagnostic or treatment decisions. Serologic testing is not useful early in the course of Lyme disease because of the low sensitivity of tests in early disease. Serologic testing may be more useful in later disease, at which time sensitivity and specificity of the test are improved. Positive or equivocal results on an ELISA, IFA, or immunodot assay requires supplemental testing with a Western blot assay. A negative result on the Western blot or ELISA indicates that there is no serologic evidence of infection by B burgdorferi at the time the sample was drawn.

PMID: 10404913 [PubMed - indexed for MEDLINE]

110. Med Microbiol Immunol. 1999 May;187(4):213-9.

Osp17, a novel immunodominant outer surface protein of Borrelia afzelii: recombinant expression in Escherichia coli and its use as a diagnostic antigen for serodiagnosis of Lyme borreliosis.

Jauris-Heipke S, Rössle B, Wanner G, Habermann C, Rössler D, Fingerle V, Lehnert G, Lobentanzer R, Pradel I, Hillenbrand B, Schulte-Spechtel U, Wilske B.

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität München, Germany.

Western blot analyses of the human humoral response of patients with Lyme borreliosis have shown that a 17-kDa protein is an immunodominant protein in late disease. Immune electron microscopy with a monoclonal antibody against this protein revealed that the 17-kDa protein is abundantly expressed on the surface of Borrelia afzelii strain PKo. Therefore, the protein has been renamed outer surface protein (Osp) 17. Recombinant Osp 17 of strain PKo was expressed in Escherichia coli and purified by chromatography. Immunoblot analysis of human sera showed a comparable sensitivity with recombinant and natural proteins. The DNA sequences of the osp17 genes from different B. afzelii strains were determined. The DNA sequences of the different osp 17 homologues (six isolates from skin, three isolates from CSF and one isolate from synovial fluid) had high sequence identities of at least 94%. Using a polyclonal antibody against recombinant Osp 17, it was shown that Osp 17 expression varied considerably among the investigated B. afzelii strains. As previously also observed for OspA- and OspC-encoding genes, the osp 17 gene is present in strains not expressing the respective protein. It has been shown that OspA and OspC expression varies in different environments such as tick and vertebrate host. Studies are underway to examine whether this is also true for Osp 17. For diagnostic purposes the use of recombinant Osp 17 has the advantage that the amount of Osp 17 antigen can be easily standardized for immunoblotting, and that this antigen can be used in a protein-specific enzyme-linked immunosorbent assay.

PMID: 10363678 [PubMed - indexed for MEDLINE]

111. Eur J Pediatr. 1998 Nov;157(11):953-4.

False-positive serological tests for Lyme disease in facial palsy and varicella zoster meningo-encephalitis.

Woelfle J, Wilske B, Haverkamp F, Bialek R.

PMID: 9835449 [PubMed - indexed for MEDLINE]

112. J Immunol Methods. 1998 Sep 1;218(1-2):9-17.

A one-step solid phase immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi.

Schønau A, Stender H, Grauballe PC.

Department of Microbiology, DAKO, Glostrup, Denmark.

A one-step immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi has been developed. The assay is based on C1q, which binds to immune complexes containing IgG and/or IgM antibodies. Micro-beads pre-coated with antibodies to human C1q are mixed with human serum samples and fluorochrome-labelled B. burgdorferi flagellum antigen. In the presence of serum IgG and/or IgM antibodies to B. burgdorferi, fluorochrome-labelled antigen/antibody complexes are formed. These are then bound by serum C1q and are subsequently captured on the anti-C1q-coated beads. The sample is analysed on a flow cytometer and the presence of fluorescent beads is, thus, indicative of a positive test result. In the present study the sensitivity and specificity of the assay are compared to those of the indirect IDEIA B. burgdorferi IgG and the mu-chain capture IDEIA B. burgdorferi IgM ELISAs for separate determination of IgG and IgM. Detection using a flow cytometer can be performed without separation of the beads from the reaction mixture, which means that in practice, the method is carried out as a one-step assay and it is, thus, very suitable for automation. Other advantages of this kind of assay includes an antibody/antigen reaction which occurs in solution and the potential of using the method for the detection of antibodies against several antigens from the same or different infectious agents (multi-parameter screening).

PMID: 9819119 [PubMed - indexed for MEDLINE]

113. J Clin Microbiol. 1998 Dec;36(12):3474-9.

Peptide-based OspC enzyme-linked immunosorbent assay for serodiagnosis of Lyme borreliosis.

Mathiesen MJ, Christiansen M, Hansen K, Holm A, Asbrink E, Theisen M.

Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark.

Sera from 210 patients with Lyme borreliosis (LB) were studied by an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (pepC10) comprising the C-terminal 10-amino-acid residues of OspC of Borrelia burgdorferi. We found that 36.3 and 45.0% of the serum samples from patients with erythema migrans (EM) and neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM) anti-pepC10 reactivities, while these samples rarely (

PMCID: PMC105224 PMID: 9817857 [PubMed - indexed for MEDLINE]

114. Rev Med Chil. 1998 Jun;126(6):702-14.

[Laboratory diagnosis of Lyme borreliosis].

[Article in Spanish]

Gutiérrez J, Núñez F, Maroto MC.

Departamento de Microbiología, Hospital Universitario San Cecilio, Universidad de Granada, España.

Some direct methods that can be used for the diagnosis of Lyme borreliosis are the culture, direct visualization or the detection of microbial DNA using polymerase chain reactions, in body tissues or fluids. Unfortunately, all these methods have a low sensitivity. There is a wide assortment of tests and antigens for indirect diagnosis, and the most recommended are ELISA tests and Western blot. The main inconvenient of these tests are the existence of shared serologic reactions, the variability of immune response and the difficult interpretation of results. Therefore, we propose the following guidelines for the diagnosis of Lyme borreliosis: For sero-epidemiological studies and to diagnose infection, antibodies should be determined in subjects with a compatible clinical picture, using an ELISA test that must be positive in at least two separate samples. All positive ELISA results should be confirmed with Western blot analysis, that must be interpreted using established criteria. Polymerase chain reactions should be used when they are available.

PMID: 9778879 [PubMed - indexed for MEDLINE]

115. Rev Med Chil. 1998 Jun;126(6):629-36.

[Efficacy of two gene species in the serological diagnosis of Borrelia burgdorferi infections].

[Article in Spanish]

Gutiérrez J, Núñez F, Piédrola G, Maroto C.

Departamento de Microbiología, Hospital Universitario San Cecilio, Universidad de Granada, España.

BACKGROUND: There is phenotypic and genetic variability among the species Borrelia burgdorferi that produces Lyme disease. Three gene species and seven serotypes have been defined. AIM: To study the efficacy of two gene species in the serological diagnosis of Borrelia burgdorferi infections in Granada, Spain. MATERIAL AND

METHODS: One thousand sixty nine sera coming from 1,251 subjects without Lyme borreliosis were analyzed. These subjects were studied for health or pregnancy controls, differential diagnosis of viral disease, diagnosis of syphilis, neurological or rheumatic diseases. In all samples, antibodies against Borrelia burgdorferi (B31 and Pko strains) and against Treponema pallidum were investigated. Screening tests (ELISA and hemagglutination) were followed by confirmations tests for positive samples (Western Blot IgG strain B31 and FTA-abs respectively). A clinical and laboratory follow up was done for subjects with positive serological tests.

RESULTS: The global rate of positive antibodies against Borrelia burgdorferi B31 was 8.31% and against the strain Pko was 0.64%. Western blot was negative in 36% of subjects with positive ELISA B31. The distribution of antibodies against the strain B31 was acute herpes virus infection in 16%, gestation in 3%, HIV infection in 6.4%, T pallidum infection in 36%, rheumatic diseases in 25%, neurological diseases in 17.5% and health controls in 7.4%. The percentage of positive Western Blot analyzes were 0.8, 2.1 and 0.4% respectively. A reversion of positive ELISA tests was observed in 6 subjects.

CONCLUSIONS: The disparity in rates of antibodies against Borrelia burgdorferi in different geographic regions may be due to differences in the serological tests used. The high rate of false positive ELISA tests underscores the need to use other serological tests.

PMID: 9778870 [PubMed - indexed for MEDLINE]

116. Psychiatr Clin North Am. 1998 Sep;21(3):693-703, viii.

The underdiagnosis of neuropsychiatric Lyme disease in children and adults.

Fallon BA, Kochevar JM, Gaito A, Nields JA.

Department of Psychiatry, Columbia University Medical Center, New York, New York, USA.

Lyme Disease has been called "The New Great Imitator," a replacement for that old "great imitator" neurosyphilis. This article reviews the numerous psychiatric and neurologic presentations found in adults and children. It then reviews the features of Lyme Disease, which makes it almost uniquely hard to diagnose, including the complexity and unreliability of serologic tests. Clinical examples follow that illustrate those presentations of this disease that mimic attention deficit hyperactivity disorder (ADHD), depression, and multiple sclerosis.

PMID: 9774805 [PubMed - indexed for MEDLINE]

117. Nervenarzt. 1998 Aug;69(8):694-7.

[Value of antibody titers for diagnosis of neuroborreliosis].

[Article in German]

Woessner R, Treib J, Haass A, Stoll M, Holzer G, Schimrigk K.

Neurologische Klinik, Universitätskliniken des Saarlandes, Homburg/Saar.

Neuroborreliosis is a very frequent subtype of infection with Borrelia burgdorferi. Because of the widely spread inapparent infections finding of diagnosis by analysis of serum antibodies is very difficult. In the years 1990-1994 the serum of 6.775 patients of the Department of Neurology in Homburg, Germany was analysed with regard to Borrelia burgdorferi specific IgG antibodies. 24% showed a positive serum titer and 20% a borderline result. 73 patients showed a specific intrathecal IgG antibody synthesis. In contrast to patients with antibodies in serum these patients showed a significant cumulation during summer. The high percentage of positive serum titers and the season independence support the assumption of widely spread inapparent infections. If a patient shows neurological symptoms the finding of serum antibodies against Borrelia burgdorferi is not sufficient for the diagnosis of Neuroborreliosis. A specific intrathecal synthesis of antibodies, is the most reliable serological indicator for Neuroborreliosis. Intrathecal synthesis usually starts three to four weeks after the first clinical symptoms.

PMID: 9757421 [PubMed - indexed for MEDLINE]

118. Eur J Clin Microbiol Infect Dis. 1998 Mar;17(3):159-66.

Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto.

Kaiser R, Rauer S.

Neurologische Klinik und Poliklinik der Albert-Ludwigs-Universität Freiburg, Germany.

The intrathecal synthesis of borrelial-specific IgM- and IgG-antibodies was studied in 67 patients with neuroborreliosis and in 14 patients with neurosyphilis (controls). Antibody concentrations in serum and in the cerebrospinal fluid were determined by an enzyme immunoassay (EIA) using, as antigens, a sonicate of Borrelia burgdorferi, the recombinant 14 kDa flagellin fragment, the outer surface protein C (22 kDa), and the high molecular mass protein p83 (83 kDa). In the sonicate EIA, IgG- and/or IgM-antibodies to Borrelia burgdorferi in serum were detected in all patients with neuroborreliosis and in 71% of patients with neurosyphilis. Intrathecal synthesis of borrelial-specific IgG- and/or IgM-antibodies was demonstrated in 82% of patients with neuroborreliosis and in 71% of patients with neurosyphilis. Immunoglobulin G- and/or IgM-antibodies in serum against any of the recombinant antigens were detected in 92% of patients with neuroborreliosis and in none of those with neurosyphilis. Intrathecal synthesis of IgG- and/or IgM-antibodies to individual recombinant antigens was demonstrated in 67% of patients with neuroborreliosis and in none of those with neurosyphilis. The sensitivity of the recombinant antigens in serum was almost equal to that of the sonicate EIA, whereas the recombinant antigens were clearly less sensitive in the estimation of the intrathecal specific immune response. It was concluded that in suspected cases of neuroborreliosis, the estimation of high specific antibodies in the recombinant EIA will be helpful in confirming the diagnosis.

PMID: 9665296 [PubMed - indexed for MEDLINE]

119. Zentralbl Bakteriol. 1998 May;287(4):347-61.

Detection of Borrelia burgdorferi in urine specimens from dogs by a nested polymerase chain reaction.

Bauerfeind R, Kreis U, Weiss R, Wieler LH, Baljer G.

Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universität Giessen, Germany.

A nested PCR (nested flagellin PCR) carrying an internal E. coli DNA control was established and compared with an in-vitro culture method for the detection of Borrelia burgdorferi in urine specimens of dogs. The predicted specific amplicon of the flagellin gene fla was generated from all cultured strains of B. burgdorferi tested (comprising three European genospecies). In contrast, all 13 strains of seven other flagellated bacterial species were negative. The PCR detection limit yielded 20 cells of B. burgdorferi per ml of double-distilled water and approx. 250 bacteria per ml of dog urine. Using the bacterial culture method, urine specimens collected from 216 dogs in Germany were all diagnosed negative for spirochetes by in-vitro culture and dark-field microscopy. In contrast, DNA of B. burgdorferi was detected in 32 specimens (14.8%) by PCR. 31 urine specimens (14.4%) showed inhibitory activity in the PCR assay. However, 94 (44%) were inhibitory in the culture assay. The majority of the PCR-positive dogs exhibited major clinical symptoms which have not been reported in the course of B. burgdorferi infection previously, e.g. cystitis (14/32 dogs) or prostatitis (5/32 dogs). Our results indicate that the analysis of urine specimens by the nested flagellin PCR is a highly valuable procedure for the diagnosis of B. burgdorferi infections in dogs.

PMID: 9638865 [PubMed - indexed for MEDLINE]

120. J Clin Microbiol. 1998 Apr;36(4):857-61.

Enzyme-linked immunosorbent assay using recombinant OspC and the internal 14-kDa flagellin fragment for serodiagnosis of early Lyme disease.

Rauer S, Spohn N, Rasiah C, Neubert U, Vogt A.

Abteilung Immunologie, Institut für Medizinische Mikrobiologie und Hygiene der Albert-Ludwigs-Universität, Freiburg, Germany.

The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo of Borrelia burgdorferi sensu stricto were expressed as recombinant proteins in Escherichia coli and were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC-14-kDa antigen ELISA). No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA. The recombinant OspC-14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract from Borrelia afzelii PKo as the antigen. According to the manufacturer's information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens. The specificity was adjusted to 95% on the basis of data for 154 healthy controls. On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%). However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%). OspC displays sequence heterogeneity of up to 40% according to the genomospecies. However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains of B. burgdorferi sensu stricto (strain GeHo) and B. afzelii (strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen. This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study. In conclusion, we suggest that the OspC-14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease.

PMCID: PMC104650 PMID: 9542898 [PubMed - indexed for MEDLINE]

121. Eur J Pediatr. 1998 Apr;157(4):304-8.

Diagnosis of paediatric Lyme arthritis using a clinical score.

Huppertz HI, Bentas W, Haubitz I, Girschick HJ, Ganser G, Thon A, Suschke HJ, Schauer-Petrowskaja C, Minden K, Schuchmann L.

Children's Hospital, University of Würzburg, Germany.

Diagnosis of Lyme arthritis (LA) in children and adolescents may be difficult due to non-specific clinical manifestations and unreliable serological tests for antibodies to Borrelia burgdorferi. In a national prospective study, 186 children with arthritis were examined in whom the attending physicians had considered the diagnosis of LA. Ultimately, LA was confirmed in 87 patients and these were compared with the remaining 99 children in whom arthritis was attributable to other causes. In comparison to patients with other causes of arthritis, patients with LA had a higher frequency of episodic arthritis and initial knee joint arthritis, reported tick bites more frequently, were older, had a lower frequency of initial arthralgias, and there were fewer large joints involved. A score was developed in a group of these patients and tested in a second group. It enabled patients with LA to be distinguished from those with other causes of arthritis: within a range from 12 to -7 points, a score of 2.5 or less excluded LA whereas 6 or more points were highly indicative of LA. If only those children with a score result between 2.5 and 6 had been tested for antibodies to B. burgdorferi, the number of tests would have been reduced by 63%. CONCLUSION: Careful analysis of clinical presentation and use of a clinical score may help in distinguishing LA from other causes of arthritis and thus reduce unnecessary and expensive testing and uninterpretable test results.

PMID: 9578966 [PubMed - indexed for MEDLINE]

122. Zentralbl Bakteriol. 1998 Mar;287(3):241-7.

European interlaboratory comparison of Lyme borreliosis serology.

Guy EC, Robertson JN, Cimmino M, Gern L, Moosmann Y, Rijpkema SG, Sambri V, Stanek G.

PHL, Singleton Hospital, Sgeti, Swansea, UK.

Serological testing for Lyme borreliosis was compared in 5 European reference laboratories with a total of 79 sera in order to determine variations in laboratory performance. A considerable range of methods were used and several laboratories employed 2 or 3 genomospecies of Borrelia burgdorferi sensu lato. No laboratory relied routinely on a single test and each weighted the significance of the findings of the various tests differently. A difference in strategy between laboratories in high and low prevalence areas was apparent in that laboratories in low prevalence areas emphasised specificity more than sensitivity and therefore produced fewer false positives, but also missed some cases. Overall agreement between the laboratories was poor and it was concluded that there is a need for a quality assurance scheme within Europe.

PMID: 9563198 [PubMed - indexed for MEDLINE]

123. Epidemiol Mikrobiol Imunol. 1997 Dec;46(4):149-54.

[Importance of the immunoblot test in the diagnosis of Lyme borreliosis].

[Article in Czech]

Honegr K, Hulínská D, Havlasová J, Dostál V.

Infekcní Klinika, Fakultní Nemocnice, Hradec Králové.

Using a commercial kit for the examination of recombinant immunoblot the authors examined sera of 85 patients with direct evidence of Borrelia burgdorferi sensu lato in serum or cerebrospinal fluid or patients with typical dermal form of borreliosis. The results were compared with results of assessment of specific antibodies by the ELISA test. The authors investigated the importance of antibody formation against different antigens in different clinical forms of the disease. The specificity of the investigated kit was higher than that of the ELISA test, the sensitivity was lower. Examination of the immunoblot was negative in 8.8% patients with direct evidence of the causal agent of Lyme borreliosis. A suitable configuration of the recombinant immunoblot for use in Europe where three genotypes of Borrelia burgdorferi sensu lato are found requires further investigation. Another problem are criteria for evaluation of positivity of the blot in Europe.

PMID: 9471305 [PubMed - indexed for MEDLINE]

124. J Clin Microbiol. 1998 Feb;36(2):427-36.

Impact of strain heterogeneity on Lyme disease serology in Europe: comparison of enzyme-linked immunosorbent assays using different species of Borrelia burgdorferi sensu lato.

Hauser U, Krahl H, Peters H, Fingerle V, Wilske B.

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität München, Munich, Germany.

For the standardization of serological tests for Lyme borreliosis (LB) in Europe, the influence of the heterogeneity of Borrelia burgdorferi sensu lato must be assessed in detail. For this study four immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISAs) with octyl-beta-D-glucopyranoside extracts of strains PKo (Borrelia afzelii), PBi (Borrelia garinii), and PKa2 and B31 (both B. burgdorferi sensu stricto) were compared. Strains PKo, PBi, and PKa2 at the passages used for antigen preparations abundantly expressed outer surface protein C (OspC), whereas strain B31 at the passage used for antigen preparation did not express OspC. Sera (all from Germany) from 222 patients with clinically defined LB of all stages, 133 blood donors, and 458 forest workers were tested. None of the forest workers had symptoms consistent with LB at the time that the samples were collected. For IgM tests, receiver operating characteristic curves demonstrated that discrimination between sera from patients and blood donors was best with strain PKo and worst with strain B31. The discriminatory abilities of the four IgG ELISAs were similar in a diagnostically reasonable specificity range (90 to 100%). More than 20% of the sera from forest workers reacted strongly in the PKo IgG ELISA (optical density value, >1.5; other assays, less than 8%). Western blots of the sera with the most discrepant ELISA results revealed almost exclusive reactivity with p17. This highly immunogenic antigen is only expressed by strain PKo. This observation might be important for the development of assays enabling discrimination between asymptomatic or previous infection and active disease.

PMCID: PMC104554 PMID: 9466753 [PubMed - indexed for MEDLINE]

125. Ann Intern Med. 1997 Dec 15;127(12):1109-23.

Laboratory evaluation in the diagnosis of Lyme disease.

Tugwell P, Dennis DT, Weinstein A, Wells G, Shea B, Nichol G, Hayward R, Lightfoot R, Baker P, Steere AC.

University of Ottawa, Ontario, Canada.

PURPOSE: To provide a qualitative evaluation of the predictive value of the laboratory diagnosis of Lyme disease and to use the resultant data to formulate guidelines for clinical diagnosis. DATA SOURCES: A MEDLINE search of English-language articles or articles with English-language abstracts published from 1982 to 1996. DATA EXTRACTION: Sensitivity, specificity, and likelihood ratios were calculated, and a random-effects model was used to combine the proportions from the eligible studies. Prespecified criteria were used to determine which studies were eligible for analysis. DATA SYNTHESIS: Laboratory testing in general is not clinically useful if the pretest probability of Lyme disease is less than 0.20 or greater than 0.80. When the pretest probability is 0.20 to 0.80, sequential testing with enzyme-linked immunosorbent assay and Western blot is the most accurate method for ruling in or ruling out the possibility of Lyme disease.

CONCLUSIONS: Laboratory testing is recommended only in patients whose pretest probability of Lyme disease is 0.20 to 0.80. If the pretest probability is less than 0.20, testing will result in more false-positive results than true-positive results; a negative test result in this situation effectively rules out the disease.

PMID: 9412316 [PubMed - indexed for MEDLINE]

126. Med Microbiol Immunol. 1997 Oct;186(2-3):145-51.

Enzyme-linked immunosorbent assays with recombinant internal flagellin fragments derived from different species of Borrelia burgdorferi sensu lato for the serodiagnosis of Lyme neuroborreliosis.

Hauser U, Wilske B.

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität München, Germany.

The serodiagnosis of early Lyme neuroborreliosis is hampered by false negative results and one of the reasons could be the heterogeneity of strains of Borrelia burgdorferi sensu lato. For this study IgG enzyme-linked immunosorbent assays (ELISAs) with recombinant internal flagellin fragments (p41/i; amino acids 129-251) derived from strains PKo (B. afzelii), B31 (B. burgdorferi sensu stricto), and PBi (B. garinii) and ELISAs with whole-cell detergent extracts of strains PKo, PKa2 (B. burgdorferi sensu stricto), and PBi were compared. Cut off absorbance values were defined by the 94th and 92th percentiles of 200 sera from blood donors. Sera from patients with clinically defined neuroborreliosis [n = 88; 41 culture proven and 47 intentionally selected by the criteria specific IgG cerebrospinal fluid/serum index > or = 2 and a serum IgG immunofluorescence assay value of < 1:64] were tested. The sensitivities of the three whole-cell detergent-extract ELISAs were similar (46.6-49.2%), whereas those of the recombinant ELISAs were highest with p41/i:PBi (57.1%) and lowest with p41/i:PKo (21.5%). A combination of the p41/i:PBi and the p41/i:B31 test was most sensitive (59.8%). The correlation of absorbance values of different assays was very high for the three whole-cell detergent-extract ELISAs, whereas the absorbance values obtained with the three recombinant tests differed considerably. The greatest differences were observed between p41/i:PKo and any of the other two recombinant antigens. The differences in immune reactivity of patients sera (due to strain heterogeneity?) seems to have more influence on the results of an assay based on a single antigen than on a whole-cell-based test.

PMID: 9403843 [PubMed - indexed for MEDLINE]

127. Eur Respir J. 1997 Jun;10(6):1356-8.

Anti-Borrelia burgdorferi immunoglobulin seroprevalence in pulmonary sarcoidosis: a negative report.

Martens H, Zöllner B, Zissel G, Burdon D, Schlaak M, Müller-Quernheim J.

Medical Hospital, Research Centre Borstel, Germany.

The aetiology of sarcoidosis is still unknown. An infectious microorganism as causal agent for this disease could not be identified, but high titres of antibodies against Borrelia burgdorferi were detected in Chinese studies implying a causality with this disease. These findings, however, could not be reproduced by other researchers. The aim of this study was, therefore, to evaluate the possible role of these spirochetes in the pathogenesis of sarcoidosis by serological examinations. Sixty sera of patients suffering from sarcoidosis were examined for anti-B. burgdorferi immunoglobulin by enzyme-linked immunosorbent assay (ELISA). ELISAs for these antibodies show a high sensitivity, but a low specificity; therefore, a specific immunoblot was used to confirm positive results. Initially, 8% of the patients were reactive in the ELISA, and 20% of these could be confirmed by immunoblot. Therefore, the prevalence for B. burgdorferi antibodies in sarcoidosis patients was 1.6%. This result did not differ significantly from the prevalence of B. burgdorferi antibodies in 1,000 regular blood donors of the city of Hamburg (7% reactive in the ELISA, 38% confirmed via immunoblot, prevalence 2.7%). The hypothesis of causality between a B. burgdorferi infection and sarcoidosis cannot be confirmed by this data.

PMID: 9192944 [PubMed - indexed for MEDLINE]

128. Mayo Clin Proc. 1997 Jun;72(6):510-4.

Clinical comparison of borreliacidal-antibody test with indirect immunofluorescence and enzyme-linked immunosorbent assays for diagnosis of Lyme disease.

Agger WA, Case KL.

Section of Infectious Disease and Microbiology, Gundersen Lutheran Medical Center, La Crosse, Wisconsin 54601, USA.

Comment in Mayo Clin Proc. 1997 Nov;72(11):1093-4.

OBJECTIVE: To compare the clinical results with the borreliacidal-antibody test (BAT) and two standard screening serologic tests for Lyme disease (LD)-the indirect immunofluorescence assay (IFA) and the enzyme-linked immunosorbent assay (ELISA). DESIGN: The medical records of patients from an endemic LD area, who had been serologically tested during the summer of 1992, were retrospectively categorized by clinical diagnoses without results of serologic tests. Serologic testing, which included control serum samples from patients from a nonendemic LD area, was performed in a blinded fashion, and the results were compared with the clinical categories. MATERIAL AND

METHODS: Medical records of 307 patients who had been serologically tested for LD were reviewed. We found untreated, active LD in 43 patients (early-localized LD, 21; early-disseminated LD, 14; and late-disseminated LD, 8) and treated LD in 33. Non-LD cases were categorized into acute or chronic conditions of unknown or known cause.

RESULTS: Overall, the BAT had a sensitivity of 11% in active LD and did not correlate with results of other conventional surface antibody assays. The IFA and ELISA were more sensitive (67 to 93%), but false-positive results frequently were noted (20 to 40%) in acute and chronic non-LD inflammatory conditions. The specificity of the BAT, IFA, and ELISA in the control group was 96%, 93%, and 97%, respectively. CONCLUSION: Until the sensitivity, as measured by prospective clinical studies, is improved without loss of specificity, the BAT should not be used clinically for the diagnosis of LD. Suspected cases of LD with atypical clinical manifestations should have positive ELISA and IFA results confirmed with a standardized immunoblot assay.

PMID: 9179134 [PubMed - indexed for MEDLINE]

129. Infect Immun. 1997 Apr;65(4):1228-36.

Complement-mediated serum sensitivity among spirochetes that cause Lyme disease.

van Dam AP, Oei A, Jaspars R, Fijen C, Wilske B, Spanjaard L, Dankert J.

Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, The Netherlands.

Borrelia burgdorferi-related isolates were tested for their sensitivity to normal human serum (NHS) and their ability to activate complement. By dark-field microscopy, electron microscopy, and subsurface plating, it was shown that exposure of a Borrelia garinii isolate to 10% or more NHS resulted in immobilization, blebbing, and killing of the spirochetes. These effects were mediated by complement, since they were not seen after heat treatment of NHS, in the presence of EDTA, or in an agammaglobulinemic serum. All seven B. garinii type 5 or 6 and all four VS116/M19 strains were serum sensitive, whereas all eight Borrelia afzelii, five of eight B. garinii type 4, and three of seven B. burgdorferi sensu stricto isolates were serum resistant. The other isolates were partially serum sensitive. Four serum-sensitive B. garinii isolates had been isolated from human cerebrospinal fluid. Most likely, activation of both the alternative pathway and the classical pathway of complement was involved, since bactericidal activity was diminished in properdin-deficient sera as well as in a C1q-depleted serum and in a C4-deficient serum. Bactericidal activity could be restored when a serum lacking C1q or C4 was mixed with a properdin-deficient serum. Isolates with various genetic backgrounds were equally able to activate C3 as measured by enzyme-linked immunosorbent assay. In the presence of Mg-EGTA, C3 was activated by all isolates after exposure to > or = 10% NHS. This study shows that B. burgdorferi-related spirochetes can be either serum sensitive or serum resistant in vitro and that this characteristic is associated with their genetic background.

PMCID: PMC175122 PMID: 9119456 [PubMed - indexed for MEDLINE]

130. Epidemiol Mikrobiol Imunol. 1997 Mar;46(1):3-8.

[Diagnosis of Lyme borreliosis with western blotting].

[Article in Czech]

Hulínská D.

Národní referencní laborator pro lymeskou boreliózu, Státní zdravotní ústav, Praha.

Spirochetes Borrelia burgdorferi sensu lato, selected as antigens for Western blot analyses were isolated from cerebrospinal (strain 192 M) and from blood (strain Kc90) and identified by means of monoclonal antibodies and the polymerase chain reaction (PCR) as B. garinii and B. afzelii. Differences between B. garinii and B. afzelii are in the genotype of the surface protein OspA and OspB, internal flagellin (Fla II) and the main extracellular protein (MEP). The reaction of polyclonal antibodies in 918 serum specimens and 180 specimens of cerebrospinal fluid was investigated in IgG and IgM immunoblots in patients with neurological symptoms, arthritis and skin manifestations suspect of Lyme borreliosis. Confirmation of the immunoenzyme ELISA reaction by means of immunoblots in the acute stage of borreliosis, in clinically obscure cases, in herpetic infection, mononucleosis and leptospirosis revealed a higher sensitivity and specificity of Western blot. Proteins of B. garinii with a molecular weight of 94, 84, 66, 60, 56, 41, 39, 33, 29, 22, 18 and 14 kDa were detected in the reaction with monoclonal antibodies and immunoglobulins of patients suffering from barreliosis. The frequency and intensity of the reaction of these antigens differed markedly in sera of patients suffering from borreliosis and sera of patients who suffered from a different infection. The external surface antigen OspA, OspB, OspC and protein with 39 kDA are significant markers of borreliosis. The most frequently detected antigens in cross reactions with immunoglobulins against other pathogens are proteins P66, P60, P41 which are dominant immunogens of all types of borrelias and moreover a humoral response to them develops in the acute stage of the disease. In arthritis and neuroborreliosis a different in IgG immunoblots was found.

PMID: 9162453 [PubMed - indexed for MEDLINE]

131. Med Microbiol Immunol. 1996 Nov;185(3):121-9.

Analysis of the human antibody response to outer surface protein C (OspC) of Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii.

Mathiesen MJ, Hansen K, Axelsen N, Halkier-Sørensen L, Theisen M.

Borrelia Laboratory, Department of Clinical Biochemistry, Statens Seruminstitut, Copenhagen S, Denmark.

The aim of this study was to determine by Western blotting (WB) the prevalence of anti-outer surface protein C (OspC) IgM and IgG antibodies in patients with Lyme borreliosis according to each of the three genospecies of Borrelia burgdorferi sensu lato. Strains of B. burgdorferi sensu stricto (MUL), B. garinii (DK 6), and B. afzelii (DK 26) served as antigen, all of which expressed abundant OspC. We examined sera from 117 patients with untreated early and late Lyme borreliosis, as well as from 100 blood donors and 29 patients with syphilis. WB results were compared with the B. burgdorferi flagellum enzyme-linked immunosorbent assay (ELISA) data. OspC from B. burgdorferi sensu stricto showed the lowest diagnostic sensitivity. OspC from B. garinii and B. afzelii performed almost identically in erythema migrans, with an IgM positive rate of 36% versus 34%, whereas OspC from B. garinii performed best in neuroborreliosis (60% versus 44%). The anti-OspC IgG response was less prominent than the IgM response and was infrequent in the late stages of the disease (0-20%). The benefit of combining the evaluation of anti-OspC responses with all three species was limited. The overall diagnostic sensitivity of WB anti-B. garinii OspC evaluation was, in the early stages of the disease, comparable to the results obtained using the flagellum ELISA. In erythema migrans and neuroborreliosis, the addition of anti-OspC IgM to the flagellum ELISA increased the sensitivity by 15% and 10%, respectively. It can, therefore, be concluded that OspC from B. garinii is a suitable OspC test antigen, and that supplementary use of OspC from other species adds little to the overall diagnostic sensitivity. An ELISA based on B. garinii OspC and native flagella seems currently the most promising concept for a future antibody test in early Lyme borreliosis.

PMID: 9007816 [PubMed - indexed for MEDLINE]

132. Infection. 1996 Nov-Dec;24(6):470-2.

Lyme borreliosis--problems of serological diagnosis.

Hofmann H.

Klinik für Dermatologie und Allergologie am Biederstein, Technische Universität München, Germany.

As long as test procedures are not standardized, the serological results of IgM- and IgG-antibodies in Lyme borreliosis must be interpreted with caution and always in the context of clinical signs and symptoms. False negative results occur primarily during the first weeks of infection. In erythema migrans of less than 4 weeks' duration, 50% of patients are seronegative even with newly designed ELISAs. At this early stage of the infection the therapeutic decision has to be established on the basis of clinical criteria. Frequently IgM- and/or IgG-antibodies develop during antibiotic therapy. After 4 weeks' duration 80% of patients have elevated borrelial antibodies detectable with recently developed ELISAs. Positive and borderline results should be confirmed by Western blot. False positive results, particularly slightly elevated IgM, may occur in a variety of other diseases. Another problem is the persistence of Borrelia-specific IgM antibodies after therapy. Serological follow-up can only be carried out with the same methods in the same laboratory. Retreatment should be considered if IgM antibodies are increasing significantly and new symptoms are occurring.

PMID: 9007597 [PubMed - indexed for MEDLINE]

133. J Clin Microbiol. 1996 Oct;34(10):2343-50.

New laboratory guidelines for serologic diagnosis of Lyme disease: evaluation of the two-test protocol.

Ledue TB, Collins MF, Craig WY.

Rheumatic Disease Laboratory, Foundation for Blood Research, Scarborough, Maine 04074, USA.

Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.

PMCID: PMC229265 PMID: 8880477 [PubMed - indexed for MEDLINE]

134. J Infect Dis. 1996 Aug;174(2):346-53.

Serodiagnosis of Lyme disease: accuracy of a two-step approach using a flagella-based ELISA and immunoblotting.

Johnson BJ, Robbins KE, Bailey RE, Cao BL, Sviat SL, Craven RB, Mayer LW, Dennis DT.

Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention Ft. Collins, Colorado 80522, USA.

An ELISA containing a purified flagellar antigen from Borrelia burgdorferi (FLA-ELISA) was evaluated. The FLA-ELISA, detecting IgM and IgG together, did not have adequate specificity by itself. Good accuracy was obtained, however, when the FLA-ELISA was the first step in a two-step protocol that used immunoblotting as a conditional second test. Samples that scored positive or equivocal by the FLA-ELISA were evaluated with separate IgM and IgG immunoblots. The sensitivity of the two-step process for patients with erythema migrans or with later manifestations of Lyme disease was 64% and 100%, respectively. The specificity for health blood donors was 100% and was 90% for the aggregate of all persons with illness that may cause serologic cross-reactivity (98% if the samples from relapsing fever patients were excluded). Test precision was 96% overall, 99% for Lyme disease case serum samples, 100% for specimens from blood donors, and 88% for samples from persons with other illness.

PMID: 8699065 [PubMed - indexed for MEDLINE]

135. Clin Infect Dis. 1996 Jul;23(1):61-5.

European Lyme borreliosis: 231 culture-confirmed cases involving patients with erythema migrans.

Strle F, Nelson JA, Ruzic-Sabljic E, Cimperman J, Maraspin V, Lotric-Furlan S, Cheng Y, Picken MM, Trenholme GM, Picken RN.

Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia.

Erratum in Clin Infect Dis 1996 Nov;23(5):1202.

In 1994, we isolated Borrelia burgdorferi sensu lato from 231 patients with erythema migrans who presented to the University Medical Center in Ljubljana, Slovenia. Samples of erythema migrans-affected skin were placed in media to support the growth of Borrelia species and evaluated in Ljubljana and Chicago. Patients whose cultures were positive included 132 women and 99 men; 136 of these 231 patients recalled a tick bite. Patients noted a rash an average of 24 days after a bite and presented a mean of 34 days after the bite with erythema migrans (mean diameter. 16 cm). Itching (44%) burning (18%), and pain (11%) were the most common local symptoms. Systemic complaints (40%) included headache, fatigue, malaise, and arthralgia. Other than erythema migrans, findings on physical examination were minimal (< 5% had fever, and in < 10% local lymph nodes were affected). Serial serological studies using indirect immunofluorescence assay, ELISA, and Western blot methods were performed, and antibodies to B, burgdorferi sensu lato were detected in < 50% of samples from patients. This is the largest series reported to date of patients with culture-confirmed Lyme borreliosis. It highlights the deficiencies of serological tests in early disease, demonstrates the sensitivity of direct detection methods for evaluation of patients with erythema migrans, and suggests that patients with early Lyme borreliosis in Slovenia may suffer a milder illness than those in the United States.

PMID: 8816130 [PubMed - indexed for MEDLINE]

136. Bioconjug Chem. 1996 May-Jun;7(3):338-42.

Presentation of peptide antigens as albumin conjugates for use in detection of serum antibodies by enzyme-linked immunosorbent assay.

Yu Z, Carter JM, Huang SY, Lackland H, Sigal LH, Stein S.

Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.

The use of linear peptides as antigens for detection of serum antibodies has been studied using a sequence of the Borrelia burgdorferi protein, flagellin, and Lyme disease sera as a model. It was found that a novel presentation of the peptide as a hapten on the carrier protein, bovine serum albumin, in the enzyme-linked immunosorbent assay format can be successfully applied to distinguish between Lyme disease and control sera.

PMID: 8816957 [PubMed - indexed for MEDLINE]

137. Med Microbiol Immunol. 1996 May;185(1):49-57.

Development of enzyme-linked immunosorbent assays using recombinant borrelial antigens for serodiagnosis of Borrelia burgdorferi infection.

Burkert S, Rössler D, Münchhoff P, Wilske B.

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität München, Germany.

To improve IgG antibody detection in the serodiagnosis of Borrelia burgdorferi infection, indirect enzyme-linked immunosorbent assays (ELISAs) were developed utilizing purified recombinant antigens of B. burgdorferi sensu lato: the chromosomally encoded proteins p100 of strain PKo (B. afzelii) and p4li (internal flagellin fragments) derived from strains PKo, PBi (B. garinii), and B31 (B. burgdorferi sensu stricto). In Western blot analysis, these proteins have proved to be highly specific and sensitive for IgG antibody detection, especially in late manifestations of Lyme borreliosis. Sera from 464 forest workers, a high-risk group for infections with B. burgdorferi, were investigated and the results compared with those obtained by a commercial ELISA using an octyl-beta-D-glucopyranoside (OGP) extract from B. afzelii (PKo) cells as the antigenic substrate. Sera derived from 200 blood donors served for determination of the 95% specific cut-off level. The number of positive test results using OGP-ELISA (23.9%) was only slightly higher than that using p100-ELISA (19.8%); corresponding results were observed in 84.7% of all sera (14.2% positive, 70.5% negative). The frequency and interassay correspondence of positive results increased with the age of the forest workers, as most markedly demonstrated by p100 and OGP assays (P < 0.0001). Using the p41i-ELISAs, generally no significant strain-dependent differences in sensitivity were found (PKo, 13.8%; PBi, 14.2%; B31, 12.9%). Compared with the p100-ELISA, the p41i-assays showed significantly lower detection rates for IgG antibodies (P < 0.03) and a reduced capacity for quantitative discrimination between seropositive individuals and negative controls (P < 0.0007). At a 95% specificity, the IgG antibody detection rate could be increased to 23.1% when the p100-ELISA was evaluated in combination with the assays using p41i/PBi and P41i/B31. Due to its high sensitivity, the specific recombinant p100-ELISA might be a suitable test for detection of late immune responses to B. burgdorferi, thus being especially useful for epidemiological investigations.

PMID: 8803953 [PubMed - indexed for MEDLINE]

138. Emerg Infect Dis. 1996 Apr-Jun;2(2):136-40.

Improved serodiagnostic testing for Lyme disease: results of a multicenter serologic evaluation.

Craven RB, Quan TJ, Bailey RE, Dattwyler R, Ryan RW, Sigal LH, Steere AC, Sullivan B, Johnson BJ, Dennis DT, Gubler DJ.

Centers for Disease Control and Prevention, Fort Collins, Colorado, USA.

PMCID: PMC2639820 PMID: 8903216 [PubMed - indexed for MEDLINE]

139. J Vet Diagn Invest. 1996 Apr;8(2):196-201.

Novel indirect fluorescent antibody test for Lyme disease.

Chambers MA, Swango LJ, Wright JC.

Department of Small Animal Internal Medicine, School of Veterinary Medicine, Tuskegee University, AL 36088, USA.

An indirect fluorescent antibody (IFA) test was developed using a novel format of Borrelia burgdorferi organisms adhered to a monolayer of cultured endothelial cells derived from an equine tumor. Sensitivity and specificity of the new IFA test for detecting anti-B, burgdorferi antibodies were evaluated using sera from dogs inoculated with live B. burgdorferi or vaccinated with B. burgdorferi bacterin or leptobacterins and from unvaccinated specific-pathogen-free (SPF) dogs. To compare the new IFA test with existing tests, serum samples were submitted to independent laboratories to be tested by enzyme-linked immunosorbent assay (ELISA) and a traditional IFA test. Samples were also tested with 2 commercially available membrane-bound ELISA kits. Both Borrelia-inoculated dogs and dogs vaccinated with B. burgdorferi bacterin developed levels of antibody detectable by the new IFA test. Dogs vaccinated with a combination canine vaccine or leptobacterin for food animal use developed detectable levels of antibody against Leptospira but remained seronegative for Borrelia by the new IFA test, as did the unvaccinated SPF dogs. The new IFA test was sensitive, detecting antibodies against B. burgdorferi as early as 7 days postinoculation. It was also specific, showing no cross-reactivity with anti-Leptospira antibodies induced by vaccination with leptobacterins. The new IFA test compared favorably with both the standardized traditional IFA test and ELISA. Results from both membrane-bound ELISA kits were not consistent when compared with each other or with the new IFA test. The new IFA test had low nonspecific fluorescence, which made it easier to evaluate and reduced the human error and variability of test results.

PMID: 8744741 [PubMed - indexed for MEDLINE]

140. J Clin Microbiol. 1996 Feb;34(2):237-40.

Use of recombinant antigens of Borrelia burgdorferi in serologic tests for diagnosis of lyme borreliosis.

Magnarelli LA, Fikrig E, Padula SJ, Anderson JF, Flavell RA.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504, USA.

Recombinant antigens of outer surface proteins (Osps) OspA, OspB, OspC, OspE, and OspF of Borrelia burgdorferi sensu stricto and of p41-G, an antigenic region of flagellin of this spirochete, were tested with human sera in class-specific and polyvalent enzyme-linked immunosorbent assays (ELISAs). In analyses for immunoglobulin M (IgM) antibodies, 18 (85.7%) of 21 serum samples from persons who had been diagnosed as having Lyme borreliosis on the basis of the presence of erythema migrans reacted positively in ELISAs with one or more Osp antigens or the p41-G antigen. Eleven serum samples contained antibodies to OspC antigen, and of these, six also reacted to the p41-G antigen and to one or more of the other recombinant antigens. The remaining five serum samples reacted solely to OspC (n = 4) or to OspC plus OspA and OspE without reactivity to p41-G (n = 1). In analyses for IgG antibodies, seropositivity was comparable to that of IgM analyses and was marked by predominant reactivity to p41-G, OspC, and OspF. Similarly, all 21 serum samples were positive in polyvalent and class-specific ELISAs with whole-cell B. burgdorferi. Minor cross-reactivity was noted when sera from persons who had syphilis, periodontitis or other oral infections, or rheumatoid arthritis were tested with OspC, OspE, OspF, and p41-G. With relatively high degrees of specificity, ELISAs with recombinant antigens, particularly OspC and p41-G, can help to confirm B. burgdorferi infections.

PMCID: PMC228775 PMID: 8788993 [PubMed - indexed for MEDLINE]

141. J Clin Microbiol. 1996 Jan;34(1):1-9.

Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans.

Aguero-Rosenfeld ME, Nowakowski J, Bittker S, Cooper D, Nadelman RB, Wormser GP.

Department of Pathology, New York Medical College, Valhalla, USA.

We investigated the appearance and evolution of immunoglobulin M (IgM) and IgG antibodies to Borrelia burgdorferi in 46 patients with culture-proven erythema migrans (EM). All patients received antimicrobial treatment and were prospectively evaluated for up to 1 year. A total of 257 serially collected serum samples were tested by commercial IgG-IgM enzyme-linked immunosorbent assay and separate IgM and IgG immunoblots (IBs). At the baseline, 33% of the patients had a positive ELISA result and 43% of the patients had a positive IgM IB result by using the criteria of the Centers for Disease Control and Prevention-Association of State and Territorial Public Health Laboratory Directors for the interpretation of IB results. Positive serology at the baseline and the rate of seroconversion correlated directly with disease duration and/or evidence of dissemination prior to treatment. At days 8 to 14 after the baseline, 91% of patients had a positive ELISA result and/or IgM IB result. Peak IgM antibody levels were seen at this time in patients with localized or disseminated disease. The most frequent IgM bands at the baseline and the peak were of 24 kDa (OspC), 41 kDa, and 37 kDa. Although 89% of the patients developed IgG antibodies as determined at a follow-up examination, only 22% were positive by the IgG IB criteria of the Centers for Disease Control and Prevention-Association of State and Territorial Public Health Laboratory Directors. The persistence of antibodies was directly related to disease duration and/or dissemination prior to treatment. Since IgM antibodies to the 24- and 41-kDa antigens remained detectable for long periods, 38% of IgM IBs were still positive at 1 year postbaseline. IgM to antigens of 39, 58, 60, 66, or 93 kDa, conversely, were most often seen in sera obtained within 1 month postbaseline. Their presence may be of assistance in confirming a recent infection with B. burgdorferi in individuals living in areas where Lyme disease is endemic.

PMCID: PMC228718 PMID: 8748261 [PubMed - indexed for MEDLINE]

142. J Clin Microbiol. 1995 Oct;33(10):2596-600.

Establishment of enzyme-linked immunosorbent assay using purified recombinant 83-kilodalton antigen of Borrelia burgdorferi sensu stricto and Borrelia afzelii for serodiagnosis of Lyme disease.

Rauer S, Kayser M, Neubert U, Rasiah C, Vogt A.

Institut für Medizinische Mikrobiologie und Hygiene, Albert-Ludwigs-Universität, Freiburg, Germany.

The 83-kDa antigen of Borrelia burgdorferi was expressed as a recombinant protein in Escherichia coli and purified for use in an enzyme-linked immunosorbent assay (p83-ELISA). Antibodies to the 83-kDa antigen of both the immunoglobulin G (IgG) and IgM isotypes could be detected in all stages of Lyme disease. Sensitivity varied, depending on the clinical stage of illness. In early stages, as defined for 118 patients with erythema migrans, it was found to be 20% (24 of 118 patients: 7 with IgM, 16 with IgG, and 1 with IgM and IgG). Of the patients with late-stage Lyme arthritis and acrodermatitis chronica atrophicans, 94% (16 of 17:2 with IgM and IgG and 14 with IgG) and 86% (36 of 42:2 with IgG and IgM and 34 with IgG) revealed positive results in the p83-ELISA, respectively. p83 displays sequence heterogeneity according to the genomospecies, but when the reactions of serum specimens from acrodermatitis chronica atrophicans patients and arthritis patients with p83 derived from representative strains of B. burgdorferi sensu stricto and Borrelia afzelii in ELISAs were compared, no differences in specificity and sensitivity were seen. When 82 serum specimens from healthy controls were tested, none had IgG and only 3 (4%) had IgM antibodies, indicating a high specificity. Positive reactions with antibodies against Treponema pallidum (1 of 37 patients; IgG) and Epstein-Barr virus (1 of 44 patients; IgM) and with autoantibodies of various specificities (1 of 53 patients; IgG) were seen with < 3% of the serum samples te11111111111111111111 high speficicity for B. burgdorferi.2+ 13% for IgM antibodies, the IgM p83-ELISA provided little diagnostic information for Lyme disease, whereas the IgG p83-ELISA appears to be a suita ;e test for serodiagnosis of advanced-stage Lyme disease.

PMCID: PMC228536 PMID: 8567889 [PubMed - indexed for MEDLINE]

143. Neurology. 1995 Sep;45(9):1663-70.

Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis.

Tumani H, Nölker G, Reiber H.

Laboratory of Neurochemistry, University of Göttingen, Germany.

We describe the specificity and sensitivity of measuring a combination of basic CSF variables and Borrelia burgdorferi (Bb)-specific IgG and IgM antibody index (AI) values for the diagnosis of early neuroborreliosis. Basic CSF variables included total cell count, quantitation of activated B cells (IgG, IgA, and IgM classes), CSF/serum quotient diagrams for IgG, IgA, and IgM (to quantitate brain-derived immunoglobulin fractions in CSF), and CSF/serum albumin ratio as a measure of blood-CSF barrier function. The Bb-specific component of immunoglobulins in CSF and serum was quantitated by ELISA. Results are based on data from CSF and serum of 24 patients with definite neuroborreliosis, 45 patients with other neurologic diseases, and 28 control individuals. Combined evidence of an elevated CSF cell count, IgM-class dominance in both the cellular and intrathecal humoral immune response, and blood-CSF barrier dysfunction yielded 70% diagnostic sensitivity and 98% diagnostic specificity for detection of neuroborreliosis. Intrathecal production of Bb-specific IgM, evaluated as Bb-specific IgM antibody index (Bb-IgM-AI; pathologic value > 1.4) showed 79% diagnostic sensitivity and 96% diagnostic specificity. Correspondingly, elevated Bb-specific IgG antibody index (Bb-IgG-AI; pathologic value > 1.4) displayed 63% diagnostic sensitivity and 89% diagnostic specificity. Combined analysis of Bb-specific AI values and basic CSF variables gave the highest sensitivity (80%) and specificity (98%). Analysis of CSF variables over a disease course showed that acute versus past disease could be discriminated by a combination of basic CSF variables and Bb-specific AI.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7675224 [PubMed - indexed for MEDLINE]

144. J Clin Microbiol. 1995 Sep;33(9):2260-4.

Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagellin, and P39 protein in patients with PCR- or culture-proven late Lyme borreliosis.

Oksi J, Uksila J, Marjamäki M, Nikoskelainen J, Viljanen MK.

Department of Medicine, Turku University Central Hospital, Finland.

The sensitivities and specificities of three enzyme-linked immunosorbent assays (ELISAs) for Borrelia burgdorferi antibodies were compared for 41 patients presenting with symptoms compatible with late Lyme borreliosis (LB) and 37 healthy controls. All subjects were living in southwestern Finland, where LB is endemic. Only patients with culture- or PCR-proven disease were enrolled in the study. The antigens of the ELISAs consisted of sonicated spirochetes, 41-kDa flagellin, and recombinant P39 protein of B. burgdorferi. Fifteen patients had strongly or moderately positive results in the serological assay(s), 19 patients had only weakly positive or borderline antibody levels, and the remaining 7 patients were seronegative by ELISA. The sensitivities of the ELISAs were 78.0% with sonicate antigen, 41.5% with 41-kDa flagellin, and 14.6% with P39 protein. The specificities of the tests were 89.2, 86.5, and 94.6%, respectively. The sonicate antigen ELISA seems to be an effective screening method. These results show that antibodies to B. burgdorferi may be present in low levels or even absent in patients with culture- or PCR-proven late LB. Therefore, in addition to serological testing, the use of PCR and cultivation is recommended in the diagnosis of LB.

PMCID: PMC228390 PMID: 7494012 [PubMed - indexed for MEDLINE]

145. Am J Med. 1995 Jul;99(1):6-12.

Bloodstream invasion in early Lyme disease: results from a prospective, controlled, blinded study using the polymerase chain reaction.

Goodman JL, Bradley JF, Ross AE, Goellner P, Lagus A, Vitale B, Berger BW, Luger S, Johnson RC.

Department of Medicine, University of Minnesota School of Medicine, Minneapolis, USA.

Erratum in Am J Med. 1996 Aug;101(2):239.

PURPOSE: The purposes of this study were to determine (1) the optimal techniques for and potential diagnostic usefulness of the polymerase chain reaction (PCR) in early Lyme disease, and (2) the true frequency and clinical correlates of PCR-documented blood-borne infection in the dissemination of Lyme disease. PATIENTS AND

METHODS: We performed a prospective, controlled, blinded study of PCR, culture, and serology on fractionated blood samples from 105 patients; 76 with physician-diagnosed erythema migrans and 29 controls. Clinical characteristics of the patients were obtained with a standardized data entry form and correlated with results of the laboratory studies.

RESULTS: Only 4 of the 76 (5.3%) patients with erythema migrans were culture positive; however, 14 of 76 (18.4%) had spirochetemia documented by PCR of their plasma. None of 29 controls were PCR or culture positive (P = 0.007, versus patients). PCR-documented spirochetemia correlated with clinical evidence of disseminated disease; 10 of 33 patients (30.3%) with systemic symptom(s) were PCR positive compared to 4 of 43 (9.3%) without such evidence (P = 0.02). PCR positivity was more frequent among patients with each of four specific symptoms: fever, arthralgia, myalgia, and headache (all P < 0.05). A higher total number of symptoms (median 2.5 in PCR-positive patients versus 0 in PCR-negative controls; P < 0.01) and the presence of multiple skin lesions (37.5% of patients with multiple, versus 13.3% of patients with single lesions [P = 0.04] were also correlated with PCR positivity. Patients with both systemic symptoms and multiple skin lesions had a 40% PCR-positivity rate; however, 4 of 42 (9.5%) asympatomatic patients with only single erythema migrans lesions were also PCR positive. In multivariate analysis using logistic regression, the number of systemic symptoms was the strongest independent predictor of PCR positivity (P = 0.004).

CONCLUSIONS: PCR detection of Borrelia burgdorferi is at least three times more sensitive than culture for identifying spirochetemia in early Lyme disease and may be useful in rapid diagnosis. PCR positivity significantly correlates with clinical evidence of disease dissemination. Bloodstream invasion is an important and common mechanism for the dissemination of the Lyme disease spirochete.

PMID: 7598144 [PubMed - indexed for MEDLINE]

146. J Rheumatol. 1995 Apr;22(4):684-8.

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

Fawcett PT, Rose CD, Gibney KM.

Department of Medical Cell Biology, Alfred I. duPont Institute, Wilmington, DE 19899, USA.

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.

PMID: 7791164 [PubMed - indexed for MEDLINE]

147. Zentralbl Bakteriol. 1995 Apr;282(3):303-14.

Intrathecal immune response in neuroborreliosis: importance of cross-reactive antibodies.

Kaiser R.

Department of Neurology, University of Freiburg, Germany.

The intrathecal IgG response to Borrelia burgdorferi was evaluated in 35 patients with neuroborreliosis (NB). Samples were tested with and without preabsorption with Treponema phagedenis. Specific antibody concentrations in the CSF and serum were determined by ELISA. The antibody index (AIBb = QBb/QIgG) was calculated from the ratio between the CSF/serum quotients for specific antibodies (QBb) and total IgG (QIgG). Intrathecal synthesis of B. burgdorferi antibodies was demonstrated in 31 samples before and in 24 samples after preabsorption with T. phagedenis. The clonal distribution of intrathecally produced IgG antibodies was determined by isoelectric focusing combined with affinity blotting. B. burgdorferi-specific oligoclonal IgG bands occurring predominantly in the CSF were demonstrated in 32/35 patients. In 29/32 samples, the major proportion of these bands also reacted with T. phagedenis. Preabsorption of samples with T. phagedenis removed a considerable share of bands reacting with B. burgdorferi. In patients with neurosyphilis, intrathecal synthesis of antibodies with specificity for B. burgdorferi was demonstrated in 7/10 samples before, and in no sample, after preabsorption of cross-reactive antibodies. Due to the lower sensitivity in determining the AIBb (-20%), preabsorption of cross-reactive antibodies cannot be generally recommended. In all patients with suspected neuroborreliosis, but uncommon neurological symptoms and missing anamnestic data concerning a tick bite or erythema migrans, neurosyphilis should be excluded by a negative TPHA test.

PMID: 7549163 [PubMed - indexed for MEDLINE]

148. J Infect Dis. 1995 Mar;171(3):724-7.

Recombinant outer surface protein C ELISA for the diagnosis of early Lyme disease.

Gerber MA, Shapiro ED, Bell GL, Sampieri A, Padula SJ.

Department of Pediatrics, University of Connecticut Health Center, Farmington.

To compare the sensitivity of a new ELISA for IgM antibodies to Borrelia burgdorferi that uses a recombinant outer surface protein C (rOspC) with those of a whole cell (WC) ELISA and an immunoblot assay for the diagnosis of early Lyme disease, serum specimens from 82 consecutive patients with physician-documented erythema migrans were analyzed. To compare the specificities of the three assays, serum specimens from 50 patients without a history of Lyme disease and from an area in which B. burgdorferi is not endemic were analyzed. The sensitivities of the WC ELISA, immunoblot assay, and IgM rOspC ELISA were 28%, 29%, and 46%, respectively, while the specificities were 100%, 100%, and 98%, respectively. The IgM rOspC ELISA is a convenient, readily automated, easily standardized serologic test that is significantly more sensitive for the diagnosis of early Lyme disease than either WC ELISA or immunoblot assay.

PMID: 7876628 [PubMed - indexed for MEDLINE]

149. J Clin Microbiol. 1995 Feb;33(2):419-27.

Immunoblot interpretation criteria for serodiagnosis of early Lyme disease.

Engstrom SM, Shoop E, Johnson RC.

Department of Microbiology, University of Minnesota Medical School, Minneapolis.

We monitored the antibody responses of 55 treated patients with early Lyme disease and physician-documented erythema migrans. Six sequential serum samples were obtained from patients before, during, and until one year after antibiotic therapy and analyzed by in-house enzyme-linked immunosorbent (ELISA) and immunoblot assays. An immunoblot procedure utilizing a gradient gel and an image analysis system was developed. A relational database management system was used to analyze the results and provide criteria for early disease immunoblot interpretation. Recommended criteria for the immunoglobulin M (IgM) immunoblot are the recognition of two of three proteins (24, 39, and 41 kDa). The recommended criteria for a positive IgG immunoblot are the recognition of two of five proteins (20, 24 [> 19 intensity units], 35, 39, and 88 kDa). Alternatively, if band intensity cannot be measured, the 22-kDa protein can be substituted for the 24-kDa protein with only a small decrease in sensitivity. Monoclonal antibodies were used to identify all these proteins except the 35-kDa protein. With the proposed immunoblot interpretations, the sequential serum samples were examined. At visit 1, the day of diagnosis and initiation of treatment, 54.5% of the serum samples were either IgM or IgG positive. The peak antibody response, with 80% of the serum samples positive, occurred at visit 2, 8 to 12 days into treatment. The sensitivities of the IgM and IgG immunoblot for detecting patients that were seropositive into the study period were 58.5 and 54.6%, respectively, at visit 1 and 100% at visit 2. Twenty percent of the patients remained seronegative throughout the study. The specificities of the IgM and IgG immunoblots were 92 to 94% and 93 to 96%, respectively. The IgM immunoblot and ELISA were similar in sensitivities, whereas the IgG immunoblot had greater sensitivity than the IgG ELISA (P = 0.006).

PMCID: PMC227960 PMID: 7714202 [PubMed - indexed for MEDLINE]

150. Wien Med Wochenschr. 1995;145(7-8):162-5.

[Growth inhibition of Borrelia burgdorferi sensu lato by antibodies. A contribution to understanding the pathogenesis and improving diagnosis of Lyme borreliosis].

[Article in German]

Sadziene A, Barbour AG.

Abteilung für Mikrobiologie und Medizin, Universität von Texas, San Antonio, USA.

Phenomenon of growth inhibition of Borrelia burgdorferi sensu lato (BBSL), the agent of Lyme disease, by antiborrelial antibodies was observed and investigated. Some of the antiborrelial antibodies were bactericidal in the absence of complement and phagocytes. Based on growth inhibition phenomenon we developed and evaluated the function-oriented in vitro growth inhibition assay (GIA). GIA proved to be more strain-specific and a better predictor of protection against infectious challenge than matrix-based assays, such as ELISA and Western blot. Growth inhibition phenomenon was also applied as a tool for selection of antibody-resistant BBSL mutants. All BBSL isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of BBSL with altered or absent Osps were selected with polyclonal or monoclonal antibodies (mAbs) at a frequency of 10(-2) to 10(-6). Based on PAGE and Western blot analysis all examined mutants were catalogued into 4 classes. Some of these mutants were later employed in studies of functional importance of Osps in immunopathogenesis of BBSL. Several studies revealed some possible functions of Osp proteins in borrelias. In one study, Osp-lacking mutant was distinguished from its Osp-bearing parent by autoaggregation and slower growth rate, diminished capacity to adhere to human umbilical vein endothelial cells, decreased plating efficiency on solid medium as well as serum and complement sensitivity. Mutant also was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant cells survived in mouse skin for the same duration as wild type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7610664 [PubMed - indexed for MEDLINE]

151. Przegl Epidemiol. 1995;49(3):257-60.

[A comparison of serologic ELISA tests from BIOMEDICA Ges. M.B.H. and VIRO-Immun Labor Diagnostica GMBH in the diagnosis of Lyme disease (introduction)].

[Article in Polish]

Kondrusik M, Daniluk J, Hermanowska-Szpakowicz T.

Klinika Chorób Pasozytniczych i Neuroinfekcji Akademii Medycznej w Białymstoku.

The purpose of this work was to evaluate detection of antibodies IgM and IgG against B. burgdorferi. We examined presence of IgM in 96 serum samples from patients suspected of Lyme disease. We looked for IgG in serum samples of 48 forest workers who submitted frequent tick bites in last two years. Among 96 patients whose sera were examined we had positive results in both tests in 5 cases and in 25 cases positive results were achieved only in test II. In 48 serum samples which we examined for presence of IgG in 2 cases positive results were in both tests, in 1 case positive in test II and negative in test I in 3 cases positive result in test I and negative in test II. According to these results we drew following initial conclusions: 1) obtained results testify better sensitivity and specificity of test II which consists of recombinant antigens of Borrelia burgdorferi, 2) no essential difference was shown in detection IgG among two used tests.

PMID: 7491420 [PubMed - indexed for MEDLINE]

152. J Clin Microbiol. 1994 Dec;32(12):2980-8.

Detection of Borrelia burgdorferi sensu lato in lesional skin of patients with erythema migrans and acrodermatitis chronica atrophicans by ospA-specific PCR.

Moter SE, Hofmann H, Wallich R, Simon MM, Kramer MD.

Institute of Immunology, University of Heidelberg, Germany.

The aim of this study was to develop a sensitive and specific PCR for the detection of Borrelia burgdorferi DNA. The plasmid-located gene coding for the outer surface protein A (OspA [31-kDa protein]) was used as a target. Nucleotide sequence information from different B. burgdorferi ospA genotypes was used to design primers homologous to different genotypes. The sensitivity of the nested PCR differed from 1 fg to 1 pg of borrelial DNA, depending on the strain analyzed. No cross-reactions with DNA from spirochetes other than B. burgdorferi or with human DNA were observed. A total of 22 skin biopsy samples from patients with erythema migrans (EM [n = 10]) or acrodermatitis chronica atrophicans (ACA [n = 12]) were examined for the presence of B. burgdorferi by nested PCR. Of 22 biopsies, 80% from EM patients and 92% from ACA patients were positive by PCR amplification. By comparison, 50% of the EM patients had elevated B. burgdorferi-specific immunoglobulin M (IgM) and/or IgG antibody levels as tested by enzyme-linked immunosorbent assay (ELISA) using purified B. burgdorferi flagella as antigen. A total of 33% of ACA patients had elevated IgM titers, and all had high IgG titers in their sera. Only 30% of specimens from patients with EM and none from patients with ACA were positive by culture. All culture-positive specimens were also positive by PCR. Thus, the sensitivities of the PCR were 80 and 92%, respectively, for patients with EM and ACA on the basis of the clinical and histopathological diagnoses of Lyme disease. From these results, we conclude that PCR is a suitable method to detect B. burdorferi sensu lato DNA in skin biopsy samples and could be applied as an additional diagnostic tool.

PMCID: PMC264211 PMID: 7883886 [PubMed - indexed for MEDLINE]

153. Infect Immun. 1994 Aug;62(8):3213-21.

Humoral immune response to outer surface protein C of Borrelia burgdorferi in Lyme disease: role of the immunoglobulin M response in the serodiagnosis of early infection.

Fung BP, McHugh GL, Leong JM, Steere AC.

Division of Rheumatology/Immunology, New England Medical Center, Boston, Massachusetts 02111.

We determined the humoral immune response to outer surface protein C (OspC) of Borrelia burgdorferi in patients with early or late manifestations of Lyme disease and investigated the use of this antigen in the serodiagnosis of early infection. The ospC gene from the low-passage human isolate 297, a North American B. burgdorferi strain, was used to make a recombinant maltose-binding protein (MBP)-OspC fusion protein for serologic tests. This gene showed 84 to 85% nucleotide sequence identity and 76 to 79% amino acid identity with ospC of B. burgdorferi B31 and 2591. The antibody responses to MBP-OspC were determined in serial sera from 15 patients with Lyme disease who were monitored for 4 to 12 years of illness, in single-serum samples from 189 patients with early or late manifestations of the disorder, and in serum samples from 106 control patients. Early in the infection, patients with erythema migrans or meningitis commonly had weak to strong immunoglobulin M (IgM) responses to OspC and sometimes weak to moderate IgG responses. Months to years later, weak to strong IgG reactivity with this protein was often apparent in patients with arthritis, but this response was weak or absent in patients with chronic neuroborreliosis. When acute- and convalescent-phase serum samples from patients with erythema migrans were tested for reactivity against MBP-OspC, the sensitivity of the IgM test was 73% and the specificity was 98%, with either enzyme-linked immunosorbent assay (ELISA) or Western blotting. We conclude that the majority of patients with Lyme disease have a prominent IgM response to OspC early in the illness, which is often followed by a prominent IgG response in patients with arthritis. For the serodiagnosis of early infection, the sensitivity and specificity of IgM ELISA and Western blotting were comparable or slightly improved when MBP-OspC was used as the antigen compared with tests in which spirochetal lysates were used.

PMCID: PMC302948 PMID: 8039891 [PubMed - indexed for MEDLINE]

154. J Clin Microbiol. 1994 Jul;32(7):1733-8.

Use of recombinant OspC from Borrelia burgdorferi for serodiagnosis of early Lyme disease.

Padula SJ, Dias F, Sampieri A, Craven RB, Ryan RW.

Department of Medicine, University of Connecticut Health Center, Farmington 06030.

Infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, is associated with an early and dominant humoral response to the spirochete's 23-kDa outer surface protein C (OspC). We have cloned and expressed OspC as a fusion protein in Escherichia coli and have shown that patient serum samples react with it in an enzyme-linked immunosorbent assay (ELISA) (S. J. Padula, A. Sampieri, F. Dias, A. Szczepanski, and R. W. Ryan, Infect. Immun. 61:5097-5105, 1993). Now we have compared the detection of B. burgdorferi-specific immunoglobulin M antibodies in 74 individuals with culture-positive erythema migrans by a whole-cell ELISA, immunoblot, and the recombinant OspC (rOspC) ELISA. Seventy-six negative controls were also studied. With all of the tests, there was a statistically significant association between the duration of disease and the frequency of a positive result. With the rOspC ELISA, the predictive value of a positive test was 100% and the predictive value of a negative test was 74%. Similar results were obtained with the whole-cell ELISA and with the immunoblot using as the source of test antigen a strain of B. burgdorferi which expresses abundant levels of OspC. We conclude that the use of rOspC in an ELISA is a convenient, readily automated, and easily standardized test for the serodiagnosis of early Lyme disease.

PMCID: PMC263779 PMID: 7929767 [PubMed - indexed for MEDLINE]

155. An Med Interna. 1994 May;11(5):212-6.

[Positive serology for Borrelia burgdorferi in patients with symptoms compatible with Lyme's disease].

[Article in Spanish]

Barranquero A, Borobio MV, Sifontes O, Martínez-Manzanares C, Izquierdo G.

Centro de Salud Polígono Sur Sevilla.

The existence of Lyme's disease (LD) in Spain has been demonstrated for several years, but the impact that LD may have on the presence of symptomatology associated to this entity has not been evaluated so far in our region. Given the existence of serological false positives and negatives, the usefulness of positive serology as indicative of LD is discussed. In this work, we try to demonstrate the presence of a greater serological positivity for anti-Bb antibodies in patients with clinical signs compatible with Lyme's disease, in order to assess in our environment if positivity of the serological tests used is indicative of infection by Bb.METHOD: In the Health Center of Pino Montano, we studied a total of 100 patients which could have been in contact with ticks or which presented symptomatology compatible with Lyme's Borreliosis, no other explantation being plausible. Determinations were made using indirect immunofluorescence (IIF) at different dilutions and three commercial ELISA methods in each serum.

RESULTS: Seropositivity for IIF at a dilution 1/128 was of 24%, similar to the one obtained with 3 different ELISA methods. This results were confirmed for each of the risk factors considered. Fever from unknown etiology was present in 33% of the cases and was associated to a higher risk of positive serology. When two systems were affected (probable LD), seropositivity was more frequent than when just one system was affected (possible LD). Advanced age in the study population was not associated to a greater risk of positive serology, the opposite of what happened in the control population.

CONCLUSIONS: The presence of anti-Bb antibodies in a risk population (probable or possible LD) is 24%, much greater than in the control population. Techniques for anti-Bb antibodies detection using ELISA methods show a significant correlation between each other and with IIF at a dilution of 1/128.

PMID: 8061134 [PubMed - indexed for MEDLINE]

156. J Clin Microbiol. 1994 May;32(5):1154-8.

Comparison of different strains of Borrelia burgdorferi sensu lato used as antigens in enzyme-linked immunosorbent assays.

Magnarelli LA, Anderson JF, Johnson RC, Nadelman RB, Wormser GP.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504.

Eight strains of Borrelia burgdorferi sensu lato were tested with serum samples from persons who had Lyme borreliosis or syphilis in class-specific enzyme-linked immunosorbent assays (ELISAs). Antigens of B. burgdorferi sensu stricto, of Borrelia garinii, and of Borrelia spirochetes in group VS461 were prepared from cultured bacteria isolated from ticks, a white-footed mouse (Peromyscus leucopus), or human tissues in North America, the former Soviet Union, and Japan. Nearly all of the serum specimens that contained immunoglobulins to strain 2591, a Connecticut isolate, were also positive in antibody tests with the other seven strains. In general, all eight strains reacted similarly and were suitable as coating antigens in class-specific ELISAs. Assay sensitivities ranged from 82.6 to 100% in analyses for immunoglobulin M and G antibodies. Compared with reference antigen strain 2591, strains 231 (a tick isolate from Canada) and NCH-1 (a human skin isolate from Wisconsin) resulted in higher antibody titers in an ELISA. Syphilitic sera cross-reacted in all tests regardless of the antigen used. Key immunodominant proteins are shared among the closely related strains of B. burgdorferi sensu lato tested, but it is suspected that variations in antigen compositions among these spirochetes may sometimes affect assay performance for detecting serum antibodies.

PMCID: PMC263628 PMID: 8051239 [PubMed - indexed for MEDLINE]

157. J Med Microbiol. 1994 Apr;40(4):293-302.

Diagnosis of Lyme borreliosis: non-specific serological reactions with Borrelia burgdorferi sonicate antigen caused by IgG2 antibodies.

Seppälä IJ, Kroneld R, Schauman K, Forsen KO, Lassenius R.

Department of Bacteriology and Immunology, University of Helsinki, Finland.

ELISA methods that measure IgG class antibodies to sonicated Borrelia burgdorferi may give false positive results. These errors could be traced to non-specific reactivity in subclass IgG2 in several instances. Sera were sampled randomly from two adult populations, which differed in having a high and low incidence of Lyme disease. If the binding of IgG2 subclass antibodies was left unrecorded in the test by the use of monoclonal reagent antibodies selective for IgG1 and IgG3, the frequency of positivity in the ELISA test decreased in samples from the low risk group. Twenty-one samples were found to be positive in an immunoblot confirmatory test. Correct prediction of positivity was obtained for 15 sera by ELISA restricted to IgG1 plus IgG3, for only four sera by ELISA restricted to IgG2 and for only six sera by IgG subclass non-restricted ELISA. A non-restricted ELISA with purified flagella of B. burgdorferi as the antigen predicted correctly 14 of the immunoblot-positive sera. The results of this ELISA correlated well with those of the IgG1 plus IgG3 subclass restricted ELISA in the high risk population (r = 0.95, prevalence of seropositivity 12%), but was significantly worse for the low risk group (r = 0.47, prevalence 2.9%). IgG subclass restriction also decreased cross-reactions of syphilitic sera in the ELISA with sonicated antigen.

PMID: 8151682 [PubMed - indexed for MEDLINE]

158. J Clin Pathol. 1994 Apr;47(4):344-9.

Predictive value of serology in diagnosing Lyme borreliosis.

Cutler SJ, Wright DJ.

Department of Medical Microbiology, Charing Cross Hospital, London.

AIMS: To compare the predictive value of immunoblotting and enzyme linked immunosorbent assay (ELISA) in diagnosing Lyme borreliosis.

METHODS: An ELISA using a whole cell sonicate of the B31 strain of Borrelia burgdorferi was used to screen samples submitted for Lyme borreliosis serology. A total of 1222 serum samples reactive in the ELISA were tested by immunoblotting also using the B31 strain. Patients with other spirochaetal diseases were tested by both methods to assess specificity, while those with erythema migrans were used to evaluate sensitivity. Subjects with different clinical conditions, which may have been associated with Lyme borreliosis, were tested using both techniques.

RESULTS: Only 16.3% of serum samples from patients submitted for Lyme borreliosis serology which were reactive by ELISA were confirmed as positive by immunoblotting. This is unlikely to represent a sensitivity problem as 51% of samples from 53 patients with erythema migrans were detected by immunoblotting compared with only 28% by ELISA. Patients whose samples were negative by ELISA were also negative by immunoblotting. Serum samples from patients with relapsing fever were reactive in both ELISA and by immunoblotting, but for other test groups immunoblotting offered increased specificity.

CONCLUSIONS: Not all ELISA results could be confirmed by immunoblotting. Yet immunoblotting was both more sensitive and specific than ELISA techniques. As a result of these observations all ELISA results should be serologically confirmed by immunoblotting. Though immunoblotting is not suited to large scale screening of samples, it can be used satisfactorily in conjunction with ELISA methods to improve the predictive value of serological tests for Lyme borreliosis.

PMCID: PMC501939 PMID: 8027373 [PubMed - indexed for MEDLINE]

159. J Clin Microbiol. 1994 Mar;32(3):777-82.

Detection of Borrelia burgdorferi in urine of Peromyscus leucopus by inhibition enzyme-linked immunosorbent assay.

Magnarelli LA, Anderson JF, Stafford KC 3rd.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504.

An inhibition enzyme-linked immunosorbent assay was developed to detect Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, in urine from white-footed mice (Peromyscus leucopus). Of the 87 urine specimens tested from 87 mice collected in widely separated tick-infested sites in Connecticut, 57 (65.5%) contained detectable concentrations of spirochetal antigens. Forty-seven (62.7%) of 75 serum samples analyzed contained antibodies to B. burgdorferi. In culture work with tissues from bladders, kidneys, spleens, or ears, 50 of 87 mice (57.5%) were infected with B. burgdorferi. Thirty-eight (76%) of 50 infected mice had antigens of this spirochete in urine, while 36 (72%) individuals had infected bladders. Of those with infected bladders, 24 (66.7%) mice excreted subunits or whole cells of B. burgdorferi into urine. Successful culturing of B. burgdorferi from mouse tissues, the presence of serum antibodies to this bacterium, and detection of antigens to this spirochete in urine provide further evidence that multiple assays can be performed to verify the presence of B. burgdorferi in P. leucopus.

PMCID: PMC263123 PMID: 8195393 [PubMed - indexed for MEDLINE]

160. Acta Neurol Scand Suppl. 1994;151:1-44.

Lyme neuroborreliosis: improvements of the laboratory diagnosis and a survey of epidemiological and clinical features in Denmark 1985-1990.

Hansen K.

Department of Infection-Immunology, Statens Seruminstitut, Copenhagen, Denmark.

Lyme neuroborreliosis (LNB) has within the last few years become one of the most frequent neuroinfections. This thesis is based on 7 publications which have two main topics: (i) to improve and develop laboratory methods for routine diagnosis of LNB, (ii) to generate epidemiological data for LNB and to achieve a descriptive clinical delimitation of this disease. Laboratory diagnosis in Lyme borreliosis is based on detection of a Borrelia burgdorferi (Bb) specific immune response. Up till now serological assays have not achieved a sufficient diagnostic specificity and sensitivity. This is partly due to the use of test antigens consisting of all proteins of the spirochete including broadly cross-reacting antigens. In order to improve diagnostic antibody detection we isolated an immunodominant structural protein of Bb, the motility organelle the flagellum. The use of native, morphologically intact flagella as test antigen in ELISA led to a significantly increased diagnostic specificity, and, especially in early disease, to an improved diagnostic sensitivity. Reservations regarding the use of only one out of the over 100 proteins of Bb as a diagnostic antigen probe are groundless. The early as well as the late immune response to Bb always includes antibodies to the flagellum. The Bb flagellum is as a test antigen not completely Bb specific. Compared with all other antigen preparations however, the flagellum is at present the best compromise, if a sensitive and specific routine serology is requested. The diagnostic performance of specific IgM detection was improved with a mu-capture ELISA, which used biotin labelled Bb flagella. Compared to conventional indirect ELISA this technique avoids false positive results due to IgM rheumatoid factor interference and false low or false negative results due to IgG competition for the test antigen. The antibody response in Bb infection develops slowly. Patients with LNB can be antibody negative in blood up to 6-8 weeks after onset of neurological symptoms. Longstanding but seronegative disease in untreated patients is unlikely to occur. Expectations of further improvement of Lyme borreliosis serology focuses presently on the performance of the outer surface protein (Osp) C as a test antigen and on the genus specific domain of the Bb flagellin. Theoretically this region constitutes the best candidate for a better test antigen either as a recombinant or a synthetic peptide. In LNB a prominent Bb specific intrathecal antibody response develops.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 8165888 [PubMed - indexed for MEDLINE]

161. J Clin Microbiol. 1993 Dec;31(12):3090-5.

Serodiagnosis in early Lyme disease.

Aguero-Rosenfeld ME, Nowakowski J, McKenna DF, Carbonaro CA, Wormser GP.

Department of Pathology, New York Medical College, Valhalla.

Erratum in J Clin Microbiol 1994 Mar;32(3):860.

Using a commercially available enzyme-linked immunosorbent assay (ELISA) and an immunoblot assay (IB), we tested sera from 100 patients with erythema migrans (EM) seen in 1991 a the Westchester County Medical Center Lyme Disease Diagnostic Center. Convalescent-phase sera were available from 59 patients. Fifty-five patients had EM of < 7 days' duration, 31 had EM of 7 to 14 days' duration, and 14 had EM of > 14 days' duration. During the acute phase of infection, 35 patients had a positive ELISA result and 43 had a positive IB result by the recently published criteria of Dressler et al. (F. Dressler, J. A. Whalen, B. N. Reinhardt, and A. C. Steere, J. Infect. Dis. 167:392-400, 1993) for interpretation of IB in patients with Lyme disease. A greater sensitivity of IB was observed in patients with EM of < 7 days' duration, as follows: 14 of 55 (25%) for IB versus 7 of 55 (13%) for ELISA (P = 0.144). Sera of all 14 patients with EM of > 14 days' duration were reactive by both tests, as follows: 13 positive and 1 equivocal by ELISA and 12 positive and 2 indeterminate by the IB. The band reactivity most frequently observed in the IB was to the 41- and 25-kDa antigens, the latter being the most frequent band observed in immunoglobulin M blots. Seroconversion was observed in 74 and 64% of evaluable patients by ELISA and IB, respectively, despite the use of antibiotic therapy.

PMCID: PMC266355 PMID: 8308100 [PubMed - indexed for MEDLINE]

162. J Med Microbiol. 1993 Oct;39(4):290-7.

Intrathecal antibody synthesis in Lyme neuroborreliosis: use of recombinant p41 and a 14-kDa flagellin fragment in ELISA.

Kaiser R, Rasiah C, Gassmann G, Vogt A, Lücking CH.

Neurologische Klinik und Poliklinik, Universität Freiburg, Germany.

The intrathecal synthesis of IgM and IgG antibodies to Borrelia burgdorferi sonicate, to recombinant flagellin (41 kDa) and to a tryptic peptide of the flagellin (14-kDa fragment) was determined by ELISA in paired cerebrospinal fluid (CSF) and serum samples from 35 patients with Lyme neuroborreliosis (LNB) and in 10 patients with neurosyphilis. The antibody index (AI = QBb/QIg) was calculated from the ratio between CSF/serum quotients for specific antibodies (QBb) and total immunoglobulins (QIg). For the examination of IgG antibodies, the sonicate ELISA was performed with and without pre-absorption with Treponema phagedenis. Of 35 patients with LNB, 31 had intrathecal IgG response to B. burgdorferi demonstrated by sonicate ELISA (24 after absorption of cross-reactive antibodies), 29 had a response demonstrated by flagellin ELISA and 21 of 35 by 14-kDa ELISA. In patients with neurosyphilis the AI (IgG) was elevated in the sonicate ELISA in 7 of 10 samples (none of 10 after absorption of cross-reactive antibodies), in the flagellin ELISA in 5 of 10 samples and in the 14-kDa ELISA in none of 10 samples. Intrathecal synthesis of IgM antibodies to B. burgdorferi was demonstrated in patients with neuroborreliosis by sonicate ELISA in 20 of 35 samples, by flagellin ELISA in 16 of 35 samples and by 14-kDa ELISA in 9 of 35 samples. No intrathecal synthesis of B. burgdorferi-specific IgM could be detected by any assay in patients with neurosyphilis.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 8411090 [PubMed - indexed for MEDLINE]

163. Enferm Infecc Microbiol Clin. 1993 Oct;11(8):460-1.

[Are seroprevalence studies useful in Lyme disease?].

[Article in Spanish]

Díaz-Míguez D, Fernández-Fernández F, Sesma P, Rodríguez-Mayo MD.

PMID: 8260522 [PubMed - indexed for MEDLINE]

164. J Neurol Sci. 1993 Aug;118(1):64-72.

Intrathecal synthesis of specific antibodies in neuroborreliosis. Comparison of different ELISA techniques and calculation methods.

Kaiser R, Lücking CH.

Department of Neurology, University of Freiburg, Germany.

The sensitivity of six different ELISA techniques and calculation methods for the determination of intrathecal synthesis of IgG antibodies specific to Borrelia burgdorferi was investigated in paired CSF and serum specimens from 33 patients with neuroborreliosis. The diagnostic value of the Antibody Index (AI) was compared with the meaningfulness of serum antibodies to B. burgdorferi (Bb), established by immunofluorescence assay (IFA). The AI, as a standard for intrathecal antibody synthesis was determined from specific antibody ratios (QBb) in the CSF and serum and the CSF/serum ratio of IgG (QIgG) or albumin (QAlb). Using Western blotting with identical concentrations of IgG in the CSF and serum all patients displayed intrathecal synthesis of specific antibodies to at least two B. burgdorferi proteins. The different ELISA methods and calculation procedures were almost equivalent in demonstrating intrathecal synthesis of specific antibodies (32 and 33/33). Calculation of AI from IFA titers was somewhat less sensitive (30/33). In 5 patients titers of serum IgG- and IgM-antibodies to B. burgdorferi determined by IFA were within the normal range or borderline, while elevated AIBb values indicated an autochthonous immune response to B. burgdorferi in the CSF. In uncertain cases of neuroborreliosis calculation of AI from ELISA titers will be useful in clarifying the diagnosis.

PMID: 8229052 [PubMed - indexed for MEDLINE]

165. Neurology. 1993 Jun;43(6):1093-8.

Detection of Borrelia burgdorferi antigens in cerebrospinal fluid.

Coyle PK, Deng Z, Schutzer SE, Belman AL, Benach J, Krupp LB, Luft B.

Department of Neurology, SUNY at Stony Brook 11794.

We examined CSF for Borrelia burgdorferi antigens using antigen-capture ELISA and Western (immuno) blot. Antigen-capture ELISA was positive in 38 of 77 (49%) CSF samples obtained from neurologic patients with presumed B burgdorferi infection, compared with one of 34 (3%) CSF samples obtained from other neurologic disease controls who came from a region endemic for Lyme disease. Western immunoblot was positive for B burgdorferi antigens in 12 of 22 (55%) CSF samples from the B burgdorferi infected groups, compared with none of 11 CSF samples from the control group. CSF antigen detection should prove helpful in evaluating patients for suspected neurologic Lyme disease.

PMID: 8170548 [PubMed - indexed for MEDLINE]

166. Ann Clin Lab Sci. 1993 May-Jun;23(3):221-9.

Serological responses in Lyme disease: the influence of sex, age, and environment.

Fidelus-Gort R, Gilmour RW, Kashatus WC.

SmithKline Beecham Clinical Laboratories, Norristown, PA 19403.

In the laboratory, the serodiagnosis of Lyme disease is a difficult decision, especially in early disease. The variability in the immune response to the Borrelia burgdorferi spirochete and the lack of specificity and sensitivity of commercial assays for the detection of antibodies in early disease have contributed to the difficulty of serodiagnosis. This study examines the serological data of over 20,000 serum specimens submitted for enzyme linked immunosorbent assay (ELISA) and/or Western Blot analysis to detect IgM and IgG antibodies to the Lyme spirochete. These samples were submitted to SmithKline Beecham Clinical Laboratories in Philadelphia from the five state region of New York, Pennsylvania, New Jersey, Delaware, and Massachusetts. These areas of the northeastern United States are considered endemic for infestation with the Borrelia burgdorferi spirochete and its vector, the deer tick. Samples were examined by positivity rate for ELISA and/or Western Blot Analysis (WBA). Specimens were broken down by age (greater than or equal to 13 years and less than 12 years) sex (male versus female), State (NY, NJ, PA, DE, MA), and by month of submission. Using the established criteria of the manufacturers for a positive response, these studies demonstrate an overall positivity rate for ELISA testing at 5.2 percent, while WBA alone had a positivity rate of 1.6 percent. Specimens examined by both ELISA and WBA had a positivity rate of 1.0 percent. Females > or = 13 years of age had the highest positivity rate of 8.5 percent on ELISA testing, while females < or = 12 years gave a positive reaction in 2.6 percent of the samples.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 8323257 [PubMed - indexed for MEDLINE]

167. J Rheumatol. 1993 Apr;20(4):734-8.

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

Fawcett PT, Rose C, Gibney KM, Chase CA, Kiehl B, Doughty RA.

Immunology Research Program, Alfred I. duPont Institute, Wilmington, DE 19899.

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.

PMID: 8496875 [PubMed - indexed for MEDLINE]

168. Am J Vet Res. 1993 Mar;54(3):398-405.

Association between clinical lameness and Borrelia burgdorferi antibody in dairy cows.

Wells SJ, Trent AM, Robinson RA, Knutson KS, Bey RF.

Department of Clinical Science, College of Veterinary Medicine, University of Minnesota, St Paul 55108.

Results of an ELISA, indirect fluorescent antibody (IFA) test, and immunoblot analysis (western blotting) for antibody to Borrelia burgdorferi in a sample of 216 lactating dairy cows were compared. The microscopic microtitration agglutination test for antibody to 6 serovars of Leptospira interrogans was also performed to evaluate possible cross-reactivity between B burgdorferi and L interrogans. Using western blotting as the standard test against which the ELISA and IFA test were compared, the ELISA had greater sensitivity (50% in summer and 38% in spring) with similar specificity (83 and 82%), compared with the IFA test (sensitivity, 6 and 5%; specificity, 90 and 83%). In addition, seropositivity to B burgdorferi, using the ELISA, was not found to be associated with seropositivity to L interrogans serovars. A matched case-control study evaluating the association between clinical lameness and antibody to B burgdorferi was performed in lactating dairy cows of 17 Minnesota and Wisconsin herds. Sera from case and control cows matched by herd, parity, and stage of lactation were evaluated, using an ELISA for B burgdorferi antibody during 2 seasons. High B burgdorferi antibody values were associated with clinical lameness in dairy cows (P = 0.006 in summer and P = 0.04 in spring).

PMID: 8498742 [PubMed - indexed for MEDLINE]

169. J Infect Dis. 1993 Feb;167(2):392-400.

Western blotting in the serodiagnosis of Lyme disease.

Dressler F, Whalen JA, Reinhardt BN, Steere AC.

Division of Rheumatology/Immunology, Tufts University School of Medicine, New England Medical Center, Boston, Massachusetts 02111.

Comment in J Infect Dis. 1993 Oct;168(4):1073.

There are currently no accepted criteria for positive Western blots in Lyme disease. In a retrospective analysis of 225 case and control subjects, the best discriminatory ability of test criteria was obtained by requiring at least 2 of the 8 most common IgM bands in early disease (18, 21, 28, 37, 41, 45, 58, and 93 kDa) and by requiring at least 5 of the 10 most frequent IgG bands after the first weeks of infection (18, 21, 28, 30, 39, 41, 45, 58, 66, and 93 kDa). When these definitions were tested in a prospective study of all 237 patients seen in a diagnostic Lyme disease clinic during a 1-year period and in 74 patients with erythema migrans or summer flu-like illnesses, the IgM blot in early disease had a sensitivity of 32% and a specificity of 100%; the IgG blot after the first weeks of infection had a sensitivity of 83% and a specificity of 95%. Among patients with indeterminate IgG responses by ELISA, 6 of 9 patients with active Lyme disease had positive blots compared with 2 of 34 patients with other illnesses (P < .001). Thus, Western blotting can be used to increase the specificity of serologic testing in Lyme disease.

PMID: 8380611 [PubMed - indexed for MEDLINE]

170. J Clin Microbiol. 1992 Dec;30(12):3158-62.

Comparison of whole-cell antibodies and an antigenic flagellar epitope of Borrelia burgdorferi in serologic tests for diagnosis of Lyme borreliosis.

Magnarelli LA, Fikrig E, Berland R, Anderson JF, Flavell RA.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504.

A recombinant protein (p41-G) of an antigenic region of flagellin was used in a standard and amplified enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Borrelia burgdorferi, the causative agent of Lyme borreliosis. Comparable sensitivities (88 to 94%) were noted when sera from 17 persons who had erythema migrans and antibodies to whole-cell B. burgdorferi were tested against the p41-G antigen. In tests of a second study group of 36 persons who had erythema migrans but no detectable antibodies to whole-cell B. burgdorferi, 3 (8%) were positive when the p41-G antigen was used. Assay specificity likewise increased when the p41-G fragment was included in an ELISA with human sera containing treponemal antibodies. Recombinant flagellar proteins of B. burgdorferi, such as the p41-G antigen, can be used in an ELISA and may help confirm Lyme borreliosis during early stages of infection and improve specificity.

PMCID: PMC270607 PMID: 1280650 [PubMed - indexed for MEDLINE]

171. Schweiz Med Wochenschr. 1992 Nov 21;122(47):1779-91.

[Immunology and diagnostic test results in Lyme borreliosis].

[Article in German]

Satz N.

Orthopädische Universitätsklinik Balgrist, Zürich.

Borrelia burgdorferi (B. burgdorferi), the etiologic agent of Lyme borreliosis, shows both a variety of outer surface proteins with molecular weights between 16 and 100 kiloDalton (kD) and a 41 kD flagellar protein, which induce the immunologic response. Lipopolysaccharides, another constituent of the bacterial capsule, are responsible for the inflammatory reaction, constitutional symptoms and for the Jarisch-Herxheimer reaction. First a vigorous T-cell immune response develops, followed later by a more slowly evolving humoral B-cell immune response. The delayed onset of the humoral immune response may explain why antibodies against B. burgdorferi could not be detected early in the course of the disease. But the antibody titers increase with duration of the illness. The humoral response shows the usual pattern of IgM appearing first, followed by IgG and IgA. The IgM titer normalizes after recovery while the IgG titer could persist over years or decades. It is hardly possible to detect B. burgdorferi microscopically or by cultivation from blood, joint or cerebrospinal fluid. For routine diagnosis the fluorescent antibody staining and the ELISA methods are available which detect IgM or IgG antibodies against B. burgdorferi. But the sensitivity and specificity of these tests are still unsatisfactory. Other methods such as the ELISA-capture method, complement binding reaction, passive hemagglutination or the polymerase chain reaction are not yet established for routine purposes. Western blot analysis did not yield an essential diagnostic advantage but may be helpful in long term observation or in special cases. The measurement of the cellular immune response by T-cell proliferation tests remains controversial. First of all Lyme borreliosis has to be diagnosed by clinical findings and by elimination through differential diagnosis. An elevated antibody titer or a positive T-cell proliferation test may confirm the diagnosis but cannot prove it. Without consistent clinical findings they are of no practical significance. Some general guidelines for interpretation of laboratory results are given.

PMID: 1448684 [PubMed - indexed for MEDLINE]

172. J Clin Epidemiol. 1992 Nov;45(11):1229-36.

Diagnostic tools in Lyme borreliosis: clinical history compared with serology.

Blaauw I, Nohlmans L, van den Bogaard T, van der Linden S.

Department of Internal Medicine, University Hospital Maastricht, The Netherlands.

The occurrence of a history of clinical Lyme borreliosis and the prevalence of positive antibodies to Borrelia burgdorferi were studied in 431 Dutch hunters. The majority of the hunters (336 or 78%) did not report any complaints and had no positive IgG antibodies to B. burgdorferi. Sixty-five hunters (15.1%) had no clinical manifestations but did not have positive antibodies to B. burgdorferi. Only 1.9% of the population studied had had past symptoms of definite or probable Lyme borreliosis. Likelihood ratios were high (21.3) for the recognition of erythema migrans, but much lower for tick bites (3.6) or positive IgG Lyme serology (3.5). Clinical history turned out to be a more powerful diagnostic tool than Lyme serology.

PMID: 1432003 [PubMed - indexed for MEDLINE]

173. J Clin Microbiol. 1992 Sep;30(9):2456-61.

Surface immunofluorescence assay for diagnosis of Lyme disease.

Cevenini R, Sambri V, Massaria F, Franchini R, D'Antuono A, Borda G, Negosanti M.

Institute of Microbiology, Policlinico S. Orsola, University of Bologna, Italy.

A surface immunofluorescence assay (SIFA) was analyzed and compared with a conventional indirect immunofluorescence assay (IFA) and whole-cell enzyme-linked immunosorbent assay (ELISA) for detecting immunoglobulin G (IgG) antibodies to Borrelia burgdorferi in sera from patients with Lyme disease. Fifty-five patients with syphilis and 33 patients with rheumatoid arthritis were used as disease controls. The sensitivity of the SIFA was low during the acute phase of Lyme disease (sera from seven of nine patients presenting with erythema chronicum migrans were negative during the first 2 months of illness); later, seroconversion was observed in all patients at various times during convalescence. Sera from five patients with complicated Lyme disease were strongly positive. SIFA was found to be highly specific, since sera from all patients with secondary or latent syphilis and patients with rheumatoid arthritis did not react in the test. Strong cross-reactivity occurred when these sera were tested in conventional IFA and ELISA; sera from 38 (69%) patients with syphilis were positive by IFA and sera from 51 (93%) patients were positive by ELISA, whereas 7 (21%) and 12 (36%) of the serum samples from patients with rheumatoid arthritis were positive by IFA and ELISA, respectively. Immunoblot analysis of SIFA-positive sera showed that the 31- and 34-kDa outer surface proteins (proteins A and B, respectively) of B. burgdorferi were the major reactive antigens involved in the test. The results support a role for SIFA in the investigation of complicated Lyme disease as well as in the differentiation of Lyme disease from other diseases associated with B. burgdorferi cross-reactive antibodies.

PMCID: PMC265523 PMID: 1401015 [PubMed - indexed for MEDLINE]

174. J Rheumatol. 1992 Apr;19(4):582-7.

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

Fawcett PT, Gibney KM, Rose CD, Dubbs SB, Doughty RA.

Immunology Laboratory, Alfred I. duPont Institute, Wilmington, Delaware 19899.

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.

PMID: 1593581 [PubMed - indexed for MEDLINE]

175. Am J Ophthalmol. 1991 Oct 15;112(4):462-3.

Prevalence of Lyme disease among patients with uveitis.

Rosenbaum JT, Rahn DW.

Comment on Am J Ophthalmol. 1989 Jan 15;107(1):77-80.

PMID: 1928255 [PubMed - indexed for MEDLINE]

176. Ann Intern Med. 1991 Oct 1;115(7):533-9.

The T-cell proliferative assay in the diagnosis of Lyme disease.

Dressler F, Yoshinari NH, Steere AC.

Tufts University School of Medicine, New England Medical Center, Boston, Massachusetts.

Comment in Ann Intern Med. 1992 Apr 1;116(7):603.

OBJECTIVE: To determine the sensitivity and specificity of the T-cell proliferative assay as a diagnostic test in Lyme disease. DESIGN: Cross-sectional study of patients with Lyme arthritis or chronic neuroborreliosis who had a history of erythema migrans, positive antibody responses to Borrelia burgdorferi by enzyme-linked immunosorbent assay (ELISA), or both; patients with other diseases; and healthy subjects. SETTING: Diagnostic Lyme disease clinic in a university hospital. PATIENTS: Forty-two of the 67 patients with active Lyme arthritis or chronic neuroborreliosis who were seen during the study period; 16 patients with inactive late Lyme disease; 77 patients with other rheumatologic or neurologic diseases; 9 workers from the Borrelia laboratory; and 9 healthy subjects. MEASUREMENTS AND MAIN

RESULTS: Nineteen of 42 patients with Lyme arthritis or chronic neuroborreliosis and 4 of 77 patients with other diseases had positive T-cell proliferative responses to B. burgdorferi antigens. The sensitivity of the proliferative assay was 45% (95% Cl, 30% to 60%) and the specificity was 95% (95% Cl, 87% to 99%). Twelve of 27 patients with active Lyme arthritis, 7 of 15 patients with chronic neuroborreliosis, 4 of 16 patients with inactive Lyme disease, 4 of 9 healthy Borrelia laboratory workers, and 0 of 9 healthy subjects had positive responses. Three of five patients with Lyme disease who had negative or indeterminant antibody responses by ELISA had positive T-cell proliferative responses. CONCLUSION: The T-cell proliferative assay may be a helpful diagnostic test in the small subset of patients with late Lyme disease who have negative or indeterminant antibody responses by ELISA.

PMID: 1883122 [PubMed - indexed for MEDLINE]

177. Arch Intern Med. 1991 Sep;151(9):1837-40.

Problems in the use of serologic tests for the diagnosis of Lyme disease.

Corpuz M, Hilton E, Lardis MP, Singer C, Zolan J.

Department of Medicine, Long Island Jewish Medical Center, Albert Einstein College of Medicine, New Hyde Park, NY 11042.

Comment in Arch Intern Med. 1992 Jun;152(6):1331.

Lyme disease can be reliably diagnosed in the presence of erythema migrans. When erythema migrans is absent, serologic tests are often used to confirm the diagnosis. To choose a test for our Lyme disease diagnostic center, serum samples were obtained from 34 patients and tested for antibodies to Borrelia burgdorferi. We evaluated five enzyme-linked immunosorbent assays from Stony Brook (NY) University Hospital, Cambridge Bioscience (Worcester, Mass), Hillcrest Biologicals (Cypress, Calif), Sigma Diagnostics (St Louis, Mo), and Zeus-Wampole Scientific Inc (Raritan, NJ) and two fluorescent antibody tests (3M [Diagnostic Systems Inc, Santa Clara, Calif] and FIAX [Whittaker M.A. Bioproducts Inc, Walkersville, Md]). A positive sample by any test was further analyzed by Western blot. Using the Centers for Disease Control (Atlanta, Ga) epidemiologic case definitions, patients were classified into those with clinical Lyme disease, patients not meeting the Centers for Disease Control definitions, and asymptomatic patients. Sensitivities of Lyme serologies varied from 13% to 73%, with Hillcrest showing the highest value and Sigma the lowest value. False-positive test results were found in 0% to 27% of patients. Western blot analysis was positive in six of 15 patients with clinical Lyme disease. These results emphasize the need for better serologic testing for Lyme disease and underline their usefulness only as adjuncts in the clinical diagnosis of Lyme disease.

PMID: 1888250 [PubMed - indexed for MEDLINE]

178. J Clin Microbiol. 1991 Sep;29(9):1761-4.

Adsorption and biotin-streptavidin amplification in serologic tests for diagnosis of Lyme borreliosis.

Magnarelli LA, Anderson JF.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504.

Serum samples from persons with Lyme borreliosis, periodontitis, or acute necrotizing ulcerative gingivitis were analyzed by an enzyme-linked immunosorbent assay (ELISA) with and without adsorption and amplification procedures. When biotin and streptavidin reagents were used as an amplification procedure in ELISA without the use of commercially prepared sorbent (Treponema phagedenis biotype Reiter), sensitivity increased. Of the 85 serum samples collected from persons with erythema migrans but no detectable antibodies to Borrelia burgdorferi by standard ELISA, 17 (20%) were reactive after amplification. Adsorption of serum samples with a 1:10 dilution of T. phagedenis biotype Reiter sorbent used in conjunction with amplified ELISA also improved the sensitivity of this method. However, cross-reactivity could not be completely eliminated. An adsorbed-amplified ELISA may be helpful in the diagnosis of Lyme borreliosis in the laboratory, particularly during early weeks of infection, when antibodies to B. burgdorferi can be present at a low concentration.

PMCID: PMC270206 PMID: 1774293 [PubMed - indexed for MEDLINE]

179. Ann Neurol. 1991 Aug;30(2):197-205.

Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi--specific immunoglobulin G, A, and M.

Hansen K, Lebech AM.

Borrelia Laboratory, Department of Infection-Immunology, Statens Seruminstitut, Copenhagen, Denmark.

An antibody capture enzyme-linked immunosorbent assay was developed to measure directly intrathecal immunoglobulin (Ig) G, A, and M synthesis specific for Borrelia burgdorferi. Purified, biotin-avidin-peroxidase-labeled B. burgdorferi flagella was used as test antigen. Paired cerebrospinal fluid and serum specimens from 100 patients with clinically definite neuroborreliosis and 35 control subjects with neurological diseases were examined. Significant B. burgdorferi-specific intrathecal IgG, A, and M production was found in 89%, 65% and 67% of patients with neuroborreliosis. Local synthesis of specific IgA was only seen in patients with significant local IgG synthesis. Antibody production in cerebrospinal fluid began by 2 weeks after onset of neurological symptoms. At the end of the second week specific IgM, IgG, or both, was detected in 88% of the patients. Specific IgG synthesis was present in all patients by 6 weeks after onset. Specific local IgM synthesis usually disappeared by 3 to 6 months after therapy, whereas specific IgG synthesis persisted after recovery. Even in patients with a severely altered blood-brain barrier, the assay discriminated between intrathecal antibody synthesis and antibody leakage from serum. The assay makes diagnostic measurement of B. burgdorferi-specific intrathecal antibody synthesis reliable, rapid, and accessible as a routine serological test.

PMID: 1897911 [PubMed - indexed for MEDLINE]

180. Enferm Infecc Microbiol Clin. 1991 Jun-Jul;9(6):335-8.

[Serologic diagnosis of Lyme disease. A pending problem].

[Article in Spanish]

Guerrero A, Quereda C, Escudero R, Cobo J, Morcillo R, Martí-Belda P.

Sección de Enfermedades Infecciosas, Hospital Ramón y Cajal, Universidad de Alcalá de Henares, Madrid.

We analyze the experience in serologic diagnosis of Lyme's borreliosis. From a total of 551 patients studied from 1987 to 1989, we further evaluate 80 cases with erythema chronicum migrans or a clinical diagnosis of Lyme's disease and positive serological tests. The techniques used were IFI, ELISA1 (Whittaker Bioproducts) and ELISA2 (MarDx Diagnostics). Serological tests results were evaluated in relation to clinical data. Five cases were excluded because of no-specific symptoms. There were 20 false-positive results, mainly due to other infections (HIV infection, tuberculosis, Mediterranean spotted fever and syphilis). Fifty-five patients were considered clinically of having Lyme's disease. IFI test was positive in 81.8% of all the 55 cases, ELISA2 in 58.4% of 53 cases tested and ELISA1 in 23% of 43 cases tested. Correlation between IFI and ELISA2 positive test was seen in 45% of cases. Specificity of all tests was higher than 97%. The study shows that sensitivity for all three techniques used was not optimal, and also there are some differences in their results. However, specificity was adequate.

PMID: 1932240 [PubMed - indexed for MEDLINE]

181. Hautarzt. 1991 Jun;42(6):356-65.

[Borrelia infections of the skin--progress of knowledge since the discovery of Lyme disease].

[Article in German]

Garbe C.

Universitäts-Hautklinik und Poliklinik, Klinikum Steglitz Freien Universität Berlin.

Comment in Hautarzt. 1992 Mar;43(3):165.

The description of Lyme disease in 1976 and the detection of its causative agent, the spirochete Borrelia burgdorferi (B. burgdorferi), in 1982 led to an increase in our knowledge of the course of B. burgdorferi infection and its clinical manifestations. The classic tick-borne dermatoses erythema chronicum migrans (ECM), lymphadenosis benigna cutis (LABC) and acrodermatitis chronica atrophicans (ACA) were proven by isolation of the spirochete from skin lesions to be caused by B. burgdorferi infection. In early disease (less than 1 year) ECM and LABC can develop locally at the site of infection (stage I), but both these skin manifestations can also occur together with multiple lesions after dissemination of the causative organism (stage II). ACA is typical for late infection (greater than 1 year, stage III). High titres of B. burgdorferi antibodies have been found in patients with localized sclerodermalike lesions (circumscribed scleroderma, lichen sclerosus et atrophicus, anetoderma), and frequent simultaneous occurrence of ACA suggests an association with late B. burgdorferi infection. Similarly, we found four cases of cutaneous B-cell lymphoma possibly arising from LABC in association with the same markers of late B. burgdorferi infection. Additionally, some cases of Schönlein-Henoch purpura and of Shulman syndrome may be associated with Lyme borreliosis. The disease is endemic in central Europe, and almost exclusively ticks of the Ixodes ricinus complex seem to transmit B. burgdorferi to humans, whereas the reservoir of infection seem to be rodents, especially mice. The main diagnostic tool is serological examination for B. burgdorferi antibodies, which will become detectable 3-6 weeks after infection. Enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence test (IFT) revealed similar sensitivity. In early disease, sensitivity for antibody detection could be improved by immunoblot technique and by flagellum-ELISA, which is specific for this early sensitizing B. burgdorferi antigen. For treatments, penicillin is no longer recommended as the drug of first choice, because low sensitivity of B. burgdorferi has been observed in vitro and in vivo. Tetracycline, doxycycline and amoxicillin p.o. are now preferred for the treatment of Lyme borreliosis, and in neurologic and cardiac abnormalities ceftriaxone i.v. is recommended. Treatment duration should be 14 days in early disease and 30 days in late disease.

PMID: 1917458 [PubMed - indexed for MEDLINE]

182. J Rheumatol. 1991 May;18(5):705-8.

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

Fawcett PT, Gibney KM, Rose CD, Klein JD, Doughty RA.

Immunology Laboratory, Alfred I. duPont Institute, Wilmington, DE 19899.

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.

PMID: 1713972 [PubMed - indexed for MEDLINE]

183. Am Clin Lab. 1991 May;10(4):14-6.

Diagnosis of Lyme disease: evaluation of an enzyme-linked immunobinding assay.

Kiefer CR.

Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.

PMID: 10148234 [PubMed - indexed for MEDLINE]

184. Scand J Infect Dis. 1991;23(1):79-87.

Use of peroxidase-labelled antigen for the detection of antibodies to Borrelia burgdorferi in human and animal sera.

Eiffert H, Lotter H, Thomssen R.

Centre of Hygiene and Human Genetics of the University, Department of Medical Microbiology, Göttingen, FRG.

We have developed a modified ELISA for the detection of anti-Borrelia burgdorferi (Bb) antibodies based on a peroxidase enzyme labelled antigen (ELAT). Microtiter plates were coated with antigen of Bb. The immunoglobulins of the serum samples were bound to the antigen and specific antibodies were detected by an enzyme labelled antigen. The test principle facilitates the recognition of specific antibodies in different collectives of human and animal sera. We performed epidemiological studies with the ELAT on 231 sera from mothers in maternity wards (9.5% positive), 219 patient sera sent to the Bb routine diagnostics (15% positive) and 230 sera from forestry workers (21.3% positive). We further investigated sera from red deer from South Lower Saxony which remained 55% Bb-antibody positive; deer were 37% and fallow deer were 29% positive.

PMID: 2028231 [PubMed - indexed for MEDLINE]

185. J Clin Microbiol. 1991 Jan;29(1):166-73.

Improved immunoglobulin M serodiagnosis in Lyme borreliosis by using a mu-capture enzyme-linked immunosorbent assay with biotinylated Borrelia burgdorferi flagella.

Hansen K, Pii K, Lebech AM.

Department of Infection Immunology, Statens Seruminstitut, Copenhagen, Denmark.

A mu-capture enzyme-linked immunosorbent assay (ELISA) for detection of serum immunoglobulin M (IgM) antibodies to Borrelia burgdorferi by using biotinylated purified B. burgdorferi flagella was developed. The diagnostic performance of the mu-capture ELISA was compared with that of a conventional indirect ELISA. Sera from untreated patients with erythema migrans (n = 50), neuroborreliosis (n = 100), and acrodermatitis chronica atrophicans (ACA; n = 48) were investigated. The cutoff of the ELISAs was adjusted to a diagnostic specificity of 98% on the basis of examination of 200 serum specimens from healthy controls. The mu-capture ELISA increased the diagnostic sensitivity in patients with erythema migrans from 32 to 48% (P less than 0.01) and in patients with neuroborreliosis from 37 to 57% (P less than 0.001). Because of an increased signal/noise ratio, the mu-capture ELISA yielded a significantly better quantitative discrimination of individual positive measurements from the cutoff (P less than 0.001). The increased signal/noise ratio was most likely a consequence of the elimination of IgG competition for the test antigen. This may also explain why 12% of patients with ACA showed significantly increased specific IgM levels only by the mu-capture ELISA. Of patients with ACA, 27% had IgM rheumatoid factor. The mu-capture principle with a directly labeled antigen showed no interference with IgM rheumatoid factor, in contrast to the indirect ELISA. The high diagnostic performance and ease of this three-step mu-capture ELISA make it suitable for routine anti-B. burgdorferi IgM serodiagnosis.

PMCID: PMC269723 PMID: 1993753 [PubMed - indexed for MEDLINE]

186. J Immunoassay. 1991;12(3):325-46.

Use of flagellin-enriched antigens in a rapid, simple and specific quantitative enzyme immunoassay for Lyme disease antibodies in human serum samples.

Lin TM, Schubert CM, Shih FF, Ahmad P, Lopez M, Horst H.

Diamedix Corporation, Miami, FL 33127.

An enzyme immunoassay (EIA, ELISA) using microwells coated with a flagellin-enriched fraction of B. burgdorferi and absorbent-containing sample diluent for the quantitative determination of Lyme disease (LD) IgG and IgM antibodies in human serum samples was described. This LD EIA required three 15-minute incubations at room temperature, followed by a 1-step normalization of photometer readings to EIA units (EU/ml). Compared with tests using the whole bacterial extract as antigens and a sample diluent containing 6% BSA, this new LD EIA revealed lower values for 20 syphilis (SS) and 21 normal serum samples (NS) but about the same for 21 Lyme disease (LD) samples, allowing lower cut-off points which would place almost all these SS and NS samples below while almost all LD samples above the positive cut-off point. The LD EIA results of larger numbers (67 to 291) of mixed samples correlated with results of four reference EIA. However, the LD EIA gave lower (2 to 4 fold) reactivities (index values) with SS and NS samples but higher values with positive serum samples than reference EIA. Thus, this LD EIA showed improvements in both specificity and sensitivity over other tests compared.

PMID: 1939664 [PubMed - indexed for MEDLINE]

187. Acta Derm Venereol. 1991;71(4):306-11.

Evaluation of the fluorescent treponemal antibody-absorption (FTA-Abs) test specificity.

Carlsson B, Hanson HS, Wasserman J, Brauner A.

Department of Bacteriology, Central Microbiological Laboratory, Stockholm County Council, Sweden.

Serum samples from 43 patients with positive test for syphilis only in the FTA-Abs test, were evaluated. Three had primary or treated syphilis. Twenty-one (49%) had clinical and/or serological signs of Lyme borreliosis as assessed by whole-cell sonicate Borrelia burgdorferi ELISA and Western blot techniques. Seven (16%) had genital Herpes simplex infection and the remaining 12 patients, miscellaneous disorders. In control sera from 30 patients with Lyme borreliosis an isolated positive FTA-Abs reaction was found in 13 patients (43%). Elevated Borrelia ELISA titres were found in nine of 30 (30%) syphilitic patient serum samples, whereas Western blots for Borrelia were negative. Six per cent of healthy blood donors were seropositive for Borrelia. Lyme borreliosis is an important cause of cross-reactions in the FTA-Abs test. Other serological tests for syphilis and Western blot for Borrelia are useful for discrimination.

PMID: 1681646 [PubMed - indexed for MEDLINE]

188. J Clin Neuroophthalmol. 1990 Dec;10(4):255-60.

The Bascom Palmer Eye Institute Lyme/syphilis survey.

Smith JL, Crumpton BC, Hummer J.

Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Florida.

Serologic screening of patients for Lyme borreliosis began at the Bascom Palmer Eye Institute (BPEI) in September 1987. This report reviews the data on 641 sera from that date up to January 1, 1990. Initially only immunofluorescent (IFA) IgG and IgM titers were obtained. Because of increasing numbers of borderline and positive IFA tests, a Lyme enzyme linked immunosorbent assay (ELISA) was added in April 1988. Also, because of significant serologic cross reactivity in patients exposed to Treponema pallidum, rapid plasma reagin (RPR) and fluorescent treponemal antibody absorption (FTA-ABS) tests were added to the serologic screening panel. Of all sera tested, 10% showed reactive RPR tests and 22% showed reactive FTA-ABS tests. Lyme IFA IgG titers were greater than or equal to 1:64 in 17% of the sera, and Lyme ELISA tests were greater than 1.25 in 15% of the sera. Our experience agrees with reports that serum RPR or VDRL tests are nonreactive in Lyme borreliosis, and that false positive FTA-ABS tests can occur in Lyme borreliosis. The importance of getting all four tests--RPR, FTA-ABS, Lyme IFA IgG and IgM, and Lyme ELISA--in all patients suspected of spirochetal disease is emphasized.

PMID: 2150843 [PubMed - indexed for MEDLINE]

189. Mol Cell Probes. 1990 Dec;4(6):451-62.

Purification of the Borrelia burgdorferi flagellum by use of a monoclonal antibody.

Fuchs R, Wilske B, Preac-Mursic V, Schierz G.

Max von Pettenkofer Institute, University of Munich, FRG.

The flagellum associated polypeptide p41 is an immunodominant antigen of Borrelia burgdorferi in the early and late stages of Lyme borreliosis. p41 was prepared by affinity chromatography using monoclonal antibody specific for p41. An immunoglobulin class specific ELISA (IgM-, IgG-ELISA) was established with purified p41 as antigen and compared to the conventional ELISA with whole cell ultrasonic antigen. Whereas the sensitivity of IgM- and IgG-ELISA was comparable in both antigen preparations, crossreactivity of sera from syphilitic patients was reduced in the p41 IgG-ELISA. Discrepant results obtained by use of ultrasonic antigen or p41 antigen, were controlled by Western blots. A correlation between the results of p41-ELISA and Western blot was shown.

PMID: 2087234 [PubMed - indexed for MEDLINE]

190. Hautarzt. 1990 Aug;41(8):424-31.

[Serodiagnosis in dermatological diseases in Borrelia burgdorferi infections].

[Article in German]

Hofmann H, Meyer-König U.

Universitäts-Hautklinik Homburg, Saar.

185 patients with dermatological symptoms of Borrelia burgdorferi infection (erythema migrans, lymphadenosis cutis benigna, acrodermatitis chronica atrophicans) and morphea were examined for Borrelia burgdorferi antibodies; in addition, sera from 173 patients were tested for exclusion of Borrelia infection. Commercially available immunofluorescence tests and enzyme immunoassays supplied by four different companies were evaluated. To investigate the specificity of these assays, sera of 34 patients with syphilis in different stages and sera from 98 control persons were examined. None of the assays evaluated was suitable for the diagnosis of early infection (sensitivity 4-35%). However, they are more reliable for the diagnosis of late infection (sensitivity 56-100%). The variation in specificity between the different assays was 82-100%. Crossreactions with Borrelia burgdorferi antibodies occurred frequently in patients with syphilis (3-47%). The reliability of serological assays should be improved by antigen purification and combination of screening and confirmatory assays. After treatment the decline in IgG and IgM antibodies is very slow.

PMID: 2272826 [PubMed - indexed for MEDLINE]

191. Am J Epidemiol. 1990 Jul;132(1):58-66.

Anti-tick antibodies: an epidemiologic tool in Lyme disease research.

Schwartz BS, Ribeiro JM, Goldstein MD.

Department of Medicine, University of Pennsylvania, Philadelphia.

In 1988, antibodies to arthropod (Ixodes dammini, Dermacentor variabilis, and Aedes aegypti) salivary gland proteins and to Borrelia burgdorferi were measured by enzyme-linked immunosorbent assay in 53 high-risk outdoor workers from the New Jersey Department of Environmental Protection. Lyme disease seropositives had significantly higher anti-I. dammini antibody levels than seronegative controls (p = 0.006). Anti-B. burgdorferi antibody (enzyme-linked immunosorbent assay) and anti-I. dammini antibody titers were highly correlated in these workers (r = 0.49, p = 0.0002). Quantitative self-reported tick exposure (tick bites in the past year) and anti-I. dammini antibody titers were significantly correlated (r = 0.27, p = 0.05), and persons without tick exposure had lower anti-I. dammini antibody levels (p = 0.009) than persons with known exposure. A second serum sample obtained 3 months after the first after a period of decreased tick exposure (the winter) revealed that the anti-I. dammini antibody levels had significantly declined (p = 0.0004). Additional experiments revealed that the antibody was not completely specific for I. dammini but was relatively specific for the two tick species examined compared with A. aegypti. The data hold promise that this antibody may be a useful tool in Lyme disease research as a biologic marker of tick exposure.

PMID: 2356814 [PubMed - indexed for MEDLINE]

192. J Clin Microbiol. 1990 Jun;28(6):1276-9.

Cross-reactivity of nonspecific treponemal antibody in serologic tests for Lyme disease.

Magnarelli LA, Miller JN, Anderson JF, Riviere GR.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504.

Serum samples obtained from 59 persons who had acute necrotizing ulcerative gingivitis, periodontitis, syphilis, or Lyme disease were tested against Treponema phagedenis biotype Reiter, Treponema denticola, Treponema vincentii, and Treponema scoliodontum by indirect fluorescent-antibody staining methods. Although there were positive reactions for sera representing each of these study groups and for 20 (13%) of 156 samples collected from the general population (premarital screening for syphilis), titration endpoints were relatively low (less than or equal to 1:256). Serum samples from 18 persons who had gingivitis or periodontitis but no history of Lyme borreliosis were tested by enzyme-linked immunosorbent assay for antibodies to Borrelia burgdorferi. Of these, five (28%) had immunoglobulin M antibody and four (22%) contained immunoglobulin G antibodies to this spirochete. Adsorption with either sorbent commercially prepared from T. phagedenis biotype Reiter or with washed, whole cells of T. phagedenis biotype Reiter reduced cross-reactivity in the enzyme-linked immunosorbent assay for Lyme borreliosis.

PMCID: PMC267918 PMID: 2380356 [PubMed - indexed for MEDLINE]

193. Diagn Microbiol Infect Dis. 1990 May-Jun;13(3):271-2.

Evaluation of three commercial tests for Lyme disease.

Cutler SJ, Wright DJ.

Department of Medical Microbiology, Charing Cross Hospital, London, England.

A commercial indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and a passive hemagglutination kit were evaluated and compared with our in-house IFA and ELISA methods. Both IFA methods gave identical results but were less sensitive than ELISA methods. Likewise, the ELISA methods were comparable. The hemagglutination method gave a statistically significant difference when compared with our ELISA method (p = 0.018 McNemar test). It is recommended that the passive hemagglutination method should not be used in its current state.

PMID: 2200637 [PubMed - indexed for MEDLINE]

194. Scand J Infect Dis Suppl. 1990;67:1-59.

Aspects of the diagnosis of Lyme borreliosis.

Karlsson M.

Department of Infectious Diseases, Danderyd Hospital, Stockholm, Sweden.

Attempts were made to culture spirochetes from the cerebrospinal fluid of 105 patients with suspected Lyme borreliosis with neurological complications. At the final evaluation, only 38 patients fulfilled the criteria of neuroborreliosis. Spirochetes were cultured from the cerebrospinal fluid of four of these patients. All four had pleocytosis in their cerebrospinal fluid and a history of neurological symptoms of only four to ten days. Two had no detectable antibodies in their cerebrospinal fluid against any of the isolated spirochetes, neither when tested with an ELISA nor by Western blot. A distinctly stronger antibody reaction to the homologous isolate than to the heterologous isolates was found in serum and cerebrospinal fluid from one patient. The cells of the isolates were morphologically similar and showed a very similar protein pattern when analyzed by SDS-PAGE. Cells of all isolates reacted with the monoclonal antibodies H5332 and H9724, which also react with Borrelia burgdorferi isolate B31, the type species. One isolate lost a major protein of 23 kD after subcultivation for four months. We conclude that isolation of spirochetes from cerebrospinal fluid is not suitable as a routine method but might prove successful in clinically selected cases of Lyme borreliosis. The patient antibody response to spirochetal components was analyzed with Western blot. Antibodies to low-molecular components including a major protein with a molecular weight of 21-23 kD, and to a 41-kD major flagellar protein, were the first to appear in serum and in CSF samples. No single band in the immunoblots was found to be specific. By requiring a 41 kD band together with at least one low-molecular band for a positive immunoblot, 53 of 68 (78%) patients with neuroborreliosis had positive IgM and/or IgG serum immunoblots by visual reading of coded material. Western blot was more sensitive than ELISA based on a sonicate antigen which identified 40 of the 68 (59%) patient samples as positive, but not significantly more sensitive than ELISA based on a purified flagellum antigen which identified 50 of 68 (74%). Western blot tended to be more sensitive than the flagellum ELISA regarding sera from patients with neurological symptoms of 2 weeks or shorter duration. However, there was a tendency towards a lower specificity regarding the serological diagnosis of current Lyme borreliosis by Western blot than by sonicate and flagellum ELISAs.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 2371553 [PubMed - indexed for MEDLINE]

195. J Clin Microbiol. 1989 Dec;27(12):2834-7.

Comparative evaluation of three products for the detection of Borrelia burgdorferi antibody in human serum.

Fister RD, Weymouth LA, McLaughlin JC, Ryan RW, Tilton RC.

University of Connecticut School of Medicine, Farmington 06032.

Eighty human serum specimens tested concomitantly by immunoblot and an enzyme-linked immunosorbent assay developed jointly at the University of Connecticut School of Medicine and the Connecticut Agricultural Experiment Station were used to evaluate three commercially available diagnostic products for Lyme borreliosis. The sources of the kits were Hillcrest Biologicals, Cypress, Calif.; Whittaker Bioproducts, Walkersville, Md.; and Cambridge Bioscience, Worcester, Mass. When compared with Western blot analysis, the sensitivities and specificities, respectively, for the diagnostic assays were as follows: Hillcrest Biologicals, 93 and 75%; Whittaker Bioproducts, 73 and 100%; Cambridge Bioscience, 89 and 100%; and University of Connecticut School of Medicine, 96 and 92%.

PMCID: PMC267136 PMID: 2687323 [PubMed - indexed for MEDLINE]

196. Rheum Dis Clin North Am. 1989 Nov;15(4):735-45.

Laboratory diagnosis of Lyme disease.

Magnarelli LA.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven.

Different techniques have been developed to aid in laboratory diagnosis of Lyme disease. The detection of serum antibodies, in particular, is relied on heavily by the medical community and is currently the most practical means of confirming B. burgdorferi infections. Although most assays may not detect low amounts of IgM antibody during the initial weeks of infection, application of a capture ELISA method has been reported to improve test sensitivity. Antibodies to Borrelia and Treponema spirochetes will cross-react in conventional assays being used for Lyme disease, but in most cases, these problems can be eliminated by performing other serologic tests or by reviewing clinical and epidemiologic data. Further improvements in the laboratory diagnosis of Lyme disease should be made by standardizing current methods (including commercial test kits), establishing reference laboratories in the United States and Europe, and by developing rapid antigen detection procedures. Finally, serologic determination of B. burgdorferi infections should remain secondary to clinical diagnosis.

PMID: 2685928 [PubMed - indexed for MEDLINE]

197. Rev Prat. 1989 May 18;39(15):1300-3.

[Lyme disease: biological diagnosis and treatment].

[Article in French]

Dournon E.

In daily practice the diagnosis of Lyme disease is confirmed in the laboratory by serological tests the specificity and sensitivity of which are not fully satisfactory. There are false-positive results due to antibodies directed against antigens others than Borrelia burgdorferi, but the main problem is that most people living in endemic areas have specific antibodies while being, and remaining, asymptomatic. In addition, the sensitivity of the current tests is mediocre at the onset of the disease. A negative serology therefore should not exclude definitively a diagnosis of Lyme disease, just as a positive serology should not compulsorily lead to this diagnosis in patients with atypical clinical signs. The treatment of Lyme disease aims at eradicating the organisms, including those wHich infest the central nervous system. Beta-lactam antibiotics seem to be particularly suitable for this purpose: amoxycillin is used in ambulatory patients, and ceftriaxone is probably the most effective treatment of severe neurological manifestations of the disease.

PMID: 2662364 [PubMed - indexed for MEDLINE]

198. J Clin Microbiol. 1989 Mar;27(3):545-51.

Serodiagnosis of erythema migrans and acrodermatitis chronica atrophicans by the Borrelia burgdorferi flagellum enzyme-linked immunosorbent assay.

Hansen K, Asbrink E.

Department of Treponematoses, Statens Seruminstitut, Copenhagen, Denmark.

The diagnostic performance of an enzyme-linked immunosorbent assay (ELISA) using purified Borrelia burgdorferi flagella as test antigen was compared with that of a B. burgdorferi sonic extract ELISA. We tested sera from 200 healthy controls, 107 patients with erythema migrans (EM), 50 patients with acrodermatitis chronica atrophicans (ACA), and 98 patients with various dermatological disorders without clinical evidence of active Lyme borreliosis. The flagellum ELISA was significantly more sensitive than the sonic extract ELISA. With sera from patients with EM, the diagnostic sensitivity for immunoglobulin G (IgG) antibody detection increased from 11.2 to 35.5% (P less than 0.001) and for IgM antibody detection it increased from 16.6 to 44.8% (P less than 0.001). In the flagellum ELISA, the number of positive tests increased significantly (P less than 0.005) when the duration of EM exceeded 1 month, but still only about 50% of patients with longstanding (1 to 12 months) untreated EM were IgG seropositive. Concomitant general symptoms did not affect the antibody level, whereas patients with multiple erythema were more frequently seropositive. All sera from patients with EM which were positive in the sonic extract ELISA were also positive in the flagellum ELISA. Not only did the overall number of positive tests increase, but the flagellum ELISA yielded a significantly better quantitative discrimination between seropositive patients and controls (P less than 0.002). IgG antibodies to the B. burgdorferi flagellum were found in all sera from patients with ACA, indicating persistence of an antiflagellum immune response in late stages of Lyme borreliosis. IgM reactivity in sera from patients with ACA was shown to be unspecific and the result IgM rheumatoid factor. A rheumatoid factor was detected in sera from 32% of patients with ACA, compared with 7.5% of patients with EM. The improved diagnostic performance, the ease of standardization of the flagellum antigen, and the lack of strain variation make the B. burgdorferi flagellum a needed reference antigen for growing routine serology in Lyme borreliosis.

PMCID: PMC267355 PMID: 2715325 [PubMed - indexed for MEDLINE]

199. J Infect Dis. 1989 Jan;159(1):43-9.

Enzyme-linked immunosorbent assays for Lyme disease: reactivity of subunits of Borrelia burgdorferi.

Magnarelli LA, Anderson JF, Barbour AG.

Department of Entomology, Connecticut Agricultural Experiment Station, New Haven 06504.

We prepared fractions of Borrelia burgdorferi, the etiologic agent of Lyme disease, from cultured spirochetes and used them as antigen in an enzyme-linked immunosorbent assay (ELISA) for IgG antibody. Polystyrene plates coated with an extract containing major proteins with apparent molecular masses of 34, 39, 59, and 68 kilodaltons had comparable sensitivity but greater specificity than plates coated with whole cells. Of the 33 serum specimens from individuals with Lyme disease that reacted with whole cells of B. burgdorferi in the class-specific ELISA, 30 (91%) remained positive when this extract was used. Cross-reactivity was minimal with antibody to treponemes. Use of subunit antigens may improve serological diagnosis of Lyme disease.

PMID: 2909642 [PubMed - indexed for MEDLINE]

200. J Clin Microbiol. 1988 Feb;26(2):338-46.

Measurement of antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease.

Hansen K, Hindersson P, Pedersen NS.

Borrelia Laboratory, Department of Treponematoses, Statens Seruminstitut, Copenhagen, Denmark.

The isolation of Borrelia burgdorferi flagella and an enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin G (IgG) and IgM to the B. burgdorferi flagellum are described. The diagnostic performance of the flagellum ELISA for serodiagnosis of Lyme disease was compared with the performance of a traditional whole cell B. burgdorferi sonic extract ELISA. We examined sera and cerebrospinal fluid (CSF) from 56 patients with lymphocytic meningoradiculitis (Bannwarth's syndrome), the most frequent secondary-stage manifestation of Lyme disease in Europe. Two hundred healthy individuals and patients with aseptic meningitis, encephalitis, Guillain-Barré syndrome, and syphilis served as controls. The flagellum ELISA was significantly more sensitive than the sonic extract ELISA. The diagnostic sensitivities were increased from 41.1 to 76.8% (P less than 0.01) for IgG and from 35.7 to 67.9% (P less than 0.05) for IgM detection in serum. The increase in sensitivity was most pronounced in patients with a short duration of disease (less than 20 days after onset). The diagnostic specificity increased for IgG detection but was almost unaltered for IgM. The flagellum ELISA did not improve the diagnostic sensitivity of measuring antibodies to borreliae in CSF, most likely owing to the low level of unspecific antibodies in CSF compared with serum. The cross-reactivity of sera and CSF from patients with syphilis decreased significantly. The flagellum antigen of B. burgdorferi shows no strain variation, is easy to purify in sufficient quantity, and is therefore a suitable reference antigen for routine serodiagnosis of Lyme disease.

PMCID: PMC266279 PMID: 3343329 [PubMed - indexed for MEDLINE]

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