Improved Sensitivity of Lyme disease Western Blots Prepared with a Mixture of <em>Borrelia Burgdorferi</em> Strains 297 and B31

Research Article

Chronic Dis Int. 2014;1(2): 7.

Improved Sensitivity of Lyme disease Western Blots Prepared with a Mixture of Borrelia Burgdorferi Strains 297 and B31

Shah JS*, Du Cruz I, Narciso W, Lo W and Harris NS

Medical Laboratory, IGeneX Inc., USA

*Corresponding author: Shah JS, IGeneX Inc., 795 San Antonio Road, Palo Alto, CA 94303, USA

Received: October 28, 2014; Accepted: December 08, 2014; Published: December 10, 2014


Testing for Borrelia burgdorferi (BB), the spirochetal agent of Lyme disease, is problematic due to poor sensitivity of commercially available serological tests. We evaluated an in-house Western Blot (WB) prepared with a mixture of two strains of BB, B31 and 297. Sensitivity and specificity of IgG and IgM testing using the two-strain WB was compared to testing with a commercial single-strain WB, and interpretation of the in-house WB using in-house criteria was compared to interpretation using criteria recommended by the Centers for Disease Control and Prevention (CDC). A total of 364 control and patient sera (including 88 from treated patients with confirmed Lyme disease) were tested. The sensitivity of the combined IgG and IgM commercial WB using CDC criteria was 77.1%. When the in-house IgG and IgM WB and CDC criteria were used, the combined sensitivity improved to 88.6 %, while use of the in-house IgG and IgM WB and in-house interpretation criteria resulted in a combined sensitivity that increased to 97.1%. Using CDC criteria, the specificity of the in-house IgG and IgM WB was 100% and 97.1%, respectively; using in-house criteria; the specificity was 95.3% and 93.1% respectively. By removal of patients who reacted to band 31kDa but tested negative for antibodies to recombinant OspA antigen, in-house IgG and IgM WB specificity increased to >97%. Based on these results, we conclude that the use of the two-strain IgG and IgM WB and in-house interpretation criteria significantly increased the combined sensitivity of the test without significant loss of specificity. In patients with indeterminate WB results, a confirmation test with recombinant OspA is recommended.

Keywords: Lyme disease; Borrelia burgdorferi; Western blot; Tick-borne disease


Borrelia Burgdorferi (BB) is the causative agent of Lyme disease – the most common tick-borne disease in North America and Europe. In Europe at least six species of BB (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, B. lusitaniae and B. spielmanii) are known to be pathogenic to humans [1,2], whereas BB sensu stricto is the only species known to cause human infections in the US [3]. The presence of a characteristic ‘bulls-eye’ Erythema Migrans (EM) rash is generally considered the earliest and best indicator of acute infection. However, the rash may be absent or may go unrecognized in 20 – 40% of the patients [4]. If the initial infection goes untreated, patients can develop disseminated Lyme disease characterized by cardiac, musculoskeletal, and neurological manifestations months to years after the initial tick bite [5-11]. Diagnosis at this stage can be even more difficult, since the history of the rash and tick bite may be lacking and the symptoms are shared with a number of other diseases [6,8,10,12].

Direct detection of the agent of Lyme disease using microscopy, culture, nucleic acid amplification and antigen detection have limited sensitivity and/or specificity, except early in the disease when an EM rash is present [4,13]. Therefore, the clinical diagnosis of Lyme disease in the US [13] and Europe [14] is usually supported by antibody detection using a two-tiered testing system. In this system, an Enzyme-linked Immunosorbent Assay (EIA) or Immunofluorescent Antibody (IFA) test is performed as a screen, followed by Western Blot (WB) confirmatory testing if the result obtained by EIA or IFA is indeterminate or positive [13]. The Centers for Disease Control and Prevention (CDC) guidelines for interpretation of the Western blot are based on the publication of Engstrom et al for IgM [15] and Dressler et al. for IgG [16], and have been the standard for WB interpretation since the Dearborn conference in1995 [13]. Two-tiered serologic testing has a reported sensitivity of 30 to 40% during the first week after presentation of the EM rash and 29 to 78% in convalescent stages after treatment [4,17]. Antibody response increases over time and the reported sensitivity in patients with neurological involvement or Lyme disease arthritis is 87% and 97% respectively [4,15,16].

Pathogens that cause diseases such as anaplasmosis, babesiosis and ehrlichiosis are transmitted by the same tick that transmits BB. Thus, Lyme disease patients may harbor these other tick-borne diseases. Therefore it is important to determine which antibodies are specific for Lyme disease [8,12,18-21]. False positive IgM and IgG results have been reported in patients with illnesses such as rheumatoid arthritis, infectious mononucleosis, autoimmune diseases, bacterial endocarditis, syphilis, other spirochetal infections and Helicobacter pylori [16,22].

The expression of different antigens depends on culture conditions and strains of BB [6,15,16]. Thus, WB assay sensitivity improves by using more than one strain of BB for preparing the WB [23,24]. Shah et al. [24] demonstrated that the IgG and IgM WB sensitivity improves without loss of specificity (1) if a mixture of two strains of BB, B31 and 297, are used for the preparation of WB strips, and (2) if WB results are considered positive when two of the following six bands are present: 23, 31, 34, 39, 41 and 93kDa.

One of the drawbacks of the two-strain WB study was that the analysis of specificity was limited to patients who were seropositive for other tick-borne diseases and healthy controls. Therefore, in the present study we evaluated 364 well-characterized specimens from healthy controls and patients with tick-borne diseases, other spirochetal infections, viral infections and autoimmune diseases. We also examined the results of WB band reactivity to: (1) determine if the in-house criteria for positive results could increase the sensitivity of the assay, and (2) the use of recombinant outer surface protein A (OspA) antigen WB could increase the WB specificity. Our goal was to provide an improved WB assay with increased sensitivity without loss of specificity.

Materials and Methods

Patient samples

A total of 364 sera including 88 sera from patients with Lyme disease were tested. The source of these sera is described in Table 1. Once received, all specimens were stored at 4°C up to one week and then at -20 °C for longer storage. Testing was performed by laboratory personnel in the same manner as clinical samples without knowledge of the expected results.