In vivo-induced antigen technology: the most sensitive method of detection for Lyme disease and other tick-borne diseases, Part One of a two-part article

Townsend Letter for Doctors and Patients, April, 2007 by Aristo Vojdani, Bernard T. Raxlen, Shirley Scott

Abstract

In search of a sensitive method for diagnosing and managing Lyme disease, we applied the commercially available Enzyme-Linked Immunosorbent Assay (ELISA), Western Blot (WB), and the newly developed In Vivo-Induced Antigen Technology, multi-peptide-based ELISA (IVIAT-MPE) to (a) 24 specimens, which, according to the College of American Pathologists (CAP) surveys, were half-positive and half-negative for Lyme disease; and (b) blood samples from 206 patients with Lyme disease. The IVIAT-MPE makes use of peptides from different Borrelia components, as well as peptides or antigens from Babesia, Ehrlichia, Bartonella, and Treponema to increase specificity.

Out of the 206 specimens, 90 (44%) tested positive by WB, and 40 as equivocal; of these 90, 87 also tested positive by IVIAT-MPE, while the other three were equivocal. Out of 40 specimens classified as equivocal by WB, 32 tested positive by IVIAT-MPE. Furthermore, out of 74 specimens tested and classified negative by WB, 25 were found positive by the IVIAT-MPE method, thereby demonstrating that IVIAT-MPE detected about 30% of specimens reported negative by ELISA or Western Blot.

Our results are as follows:

1. Demonstrated the importance of the inclusion or exclusion of Lyme disease due to overlapping symptomatologies with many different diseases

2. Showed that IVIAT is a new technique that identifies pathogen antigens that are immunogenic and are expressed in vivo during human infection

3. Showed that the measurement of antibodies against different B. burgdorferi antigens, including those induced in vivo, is an important aid in the diagnosis of Lyme disease

We therefore conclude that while correlation between WB and ELISA is good, the IVIAT-MPE is quantitative, more sensitive, species- and subspecies-specific, and detects antibodies against cross-reactive peptides or antigens from other microorganisms used in the methodology. Furthermore, due to cross-reaction with other microorganisms and antigens such as Yersinia entercolitica, Brucella, Chlamydia pneumoniae, Ricketssia ricketsii, and even arthritis-related protein (glutathione-S-transferase), immune reaction with other antigens should be excluded before implementation of long-term antibiotic treatment for Lyme disease. Based on these findings, we believe that the Multi-Peptide ELISA assay is the most sensitive technique available to confirm the diagnosis of Lyme disease.

Introduction

Lyme disease in the United States is caused by the tick-transmitted spirochete Borrelia burgdorferi sensu stricto. In Europe, Lyme borreliosis is caused primarily by infection with B. garinii and B. afzelii. (1) The illness usually begins with a characteristic, expanding skin lesion, erythema migrans (EM), which is sometimes accompanied by flu-like symptoms. (2,3) Within days to weeks, spirochetes may disseminate to other sites, particularly to the joint, the heart, and the nervous system. Depending on the immune responses of the individual, manifestations of disseminated infection such as lymphocytic meningitis, atrioventricular nodal block, or oligoarticular arthritis may develop after several months. (1,3) If untreated, the wide range of outcomes depends on the interplay between spirochetal antigenicity or virulence factors and cellular and humoral immune responses of the host. (4,5)

The diagnosis of Lyme disease is based on clinical and laboratory findings. Serologic testing is the most commonly used corroborative method, but serology also harbors several problems. The occurrence of cross-reacting antibodies may result in false-positive findings. (6-9)

Furthermore, patients may still be seronegative in the early stages of the infection, and the humoral immune response can be diminished after the early onset of antibiotic treatment. Several attempts to standardize the serological tests for Lyme disease have been done so far, (10,11) but considerable variations in test results are still present among different laboratories even when they use the same methodology. (12)

This lack of correlation between clinical symptomatology and laboratory test results is due to the antigenic diversity of Borrelia in the mammalian system. Most laboratories are using Borrelia lysate prepared from B. burgdorferi grown in the laboratory environment. (12-17) However, the use of antigens grown in culture does not represent antigenic variations and expression used by Borrelia in the mammalian system in vivo to evade the immune system and avoid immune destruction. (18) Therefore, it is crucial to combine clinical symptomatology with the most sensitive technique available to diagnose Lyme disease. (19) We used three different methods--the commercially available ELISA, Western Blot, and our own Multiple-Peptide-Based ELISA (MPE)--to test the samples obtained from patients with possible Lyme disease and from the College of American Pathologists (CAP) and compare their sensitivity and specificity.

Since the chronic nature of Lyme disease and antigenic diversity of the spirochetes suggest that antigenic variation plays an important role in immune invasion, we selected peptides from different components of Borrelia during different cycles. (20-25) These included peptides from outer surface proteins A, C, and E, leukocyte function-associated antigens, immunodominant antigens, variable major proteins, and peptides from decorin-binding proteins of Borrelial subspecies (B. sensu stricto. B. afzelii, B. garinii). (26-54) Furthermore, in the same ELISA assay, we measured antibodies against specific peptides or antigens from Babesia, Ehrlichia, Bartonella, and Treponema to confirm associated disorders or to exclude cross-reactive antibodies. (54-60)