The recent approval by FDA of an OspA vaccine is an important first step toward the prevention of Lyme borreliosis. The vaccine will likely be administered widely, especially in the upper midwestern and northeastern United States, where Lyme borreliosis is endemic. Vaccination should significantly reduce the morbidity associated with Lyme borreliosis. Unfortunately, the FDA-approved OspA vaccine was less than 50% effective in preventing infection with B. burgdorferi
sensu lato after two injections and only 78% effective after the third injection (24
). Sigal et al. (23
) also reported efficacy of only 68% after two inoculations with another OspA Lyme borreliosis vaccine which is awaiting FDA approval. Other major concerns with OspA vaccination are the duration of protective immunity (17
) and the number of booster vaccinations required to maintain sustained high levels of protective borreliacidal antibodies. There are reports of infections in humans (21
), dogs (27
), and rabbits (10
) after vaccination. These results suggest that some OspA vaccinees will become infected with B. burgdorferi
sensu lato. Accurate serodiagnosis will be severely compromised in these individuals, and the diagnostic uncertainty will be increased when patients with illness associated with a tick bite are evaluated.
Many clinical laboratories in the United States use the CDC-recommended two-tiered approach for the serodiagnosis of Lyme borreliosis recommended at the Second National Conference on the Serologic Diagnosis of Lyme Borreliosis (6
). By this approach, serum is first screened by using a sensitive ELISA or IFA. To confirm the serodiagnosis of Lyme borreliosis, equivocal and positive serum specimens are then tested by a more specific IgM and IgG WB procedure (8
). Vaccination with OspA increases the number of false-positive results by the current ELISA and IFA screening procedures because most laboratories use B. burgdorferi
sensu stricto isolates that express large amounts of OspA (2
). If the number of OspA vaccinees becomes large, the increased cost of confirmatory testing will likely become prohibitive. A screening test that can discriminate between infection with B. burgdorferi
sensu lato and vaccination is needed.
In this study, we evaluated the ability of B. burgdorferi
sensu stricto 50772 and OspC ELISAs to detect early Lyme borreliosis. B. burgdorferi
sensu stricto 50772 does not possess the ospA
) but expresses relatively high concentrations of OspC (19
). B. burgdorferi
sensu stricto 50772 and OspC were chosen because of their ability to detect anti-OspC antibodies, which are among the first antibodies detectable during early Lyme borreliosis (8
). Schwan et al. (22
) and others (26
) demonstrated that B. burgdorferi
sensu lato upregulates OspC and concomitantly downregulates OspA shortly before spirochetes are transmitted to the host. Consequently, the detection of anti-OspC antibodies should be a reliable indicator of early infection with B. burgdorferi
sensu lato, and detection should not be affected by vaccination with OspA.
We demonstrated that both B. burgdorferi
sensu stricto 50772 and OspC could be used to detect early Lyme borreliosis. Sixty-nine percent and 65% of serum samples from patients with case-defined or culture-confirmed early Lyme borreliosis had anti-B. burgdorferi
sensu stricto 50772 or anti-OspC reactivities, respectively. Magnarelli et al. (16
) and Gerber et al. (12
) also demonstrated that anti-OspC antibodies could be used to detect early Lyme borreliosis with sensitivities of 52 and 46%, respectively. Furthermore, we showed that little or no reactivity was detected in sera from individuals vaccinated with OspA when B. burgdorferi
sensu stricto 50772 or OspC was used as the detection antigen. Recently, Zhang et al. (28
) also demonstrated that an OspA-deficient B. burgdorferi
sensu lato isolate could discriminate vaccination from infection.
Despite the elimination of OspA, B. burgdorferi
sensu stricto 50772 and OspC were still highly nonspecific antigens. Similar numbers of false-positive reactions occurred with sera from healthy subjects, serum samples containing antinuclear antibodies, and sera from individuals infected with cytomegalovirus. In addition, B. burgdorferi
sensu stricto 50772 was significantly more reactive with sera from patients with syphilis and EBV infection. The mean absorbance values for sera from patients with EBV infection did not differ significantly from the absorbance values obtained with sera from patients with early Lyme borreliosis. This is likely due to polyclonal stimulation of B cells by EBV (20
). Some antibodies, for example, antibodies to the flagellar protein of B. burgdorferi
sensu lato, may cause the cross-reactivity. The cross-reactivity observed with syphilitic serum is of less concern because clinicians can differentiate patients on the basis of clinical symptoms and treponemal antibody-specific tests. However, the high degree of cross-reactivity and the inability of B. burgdorferi
sensu stricto 50772 to discriminate early Lyme disease from EBV infection may cause some confusion. Patients with EBV infection can present with clinical signs and symptoms similar to those of patients with Lyme borreliosis. Therefore, the OspC protein may be a more specific serodiagnostic antigen.
Collectively, our results and those of Zhang et al. (28
) suggest that laboratories can eliminate cross-reactivity caused by vaccination against Lyme borreliosis by modifying screening tests used in the first tier of the two-tiered approach. OspC or B. burgdorferi
sensu stricto 50772 ELISAs did not significantly react with serum from subjects vaccinated against Lyme disease. These tests also detected antibodies in over 60% of sera from patients with early Lyme borreliosis. However, both tests were highly nonspecific. The lack of specificity highlights the necessity of confirming positive findings by more specific serodiagnostic assays.
The most common confirmatory test for Lyme borreliosis is WB. It is important, however, that OspA vaccination may also increase the complexity of interpretation of WB. Most WB procedures also use OspA-expressing B. burgdorferi
sensu lato as antigen. Detection of reactivities may be obscured by OspA that does not migrate as a single band but that reacts with anti-OspA antibody from vaccinees. Another option for confirming Lyme borreliosis serodiagnostically is by detection of borreliacidal antibodies. This procedure would not be affected by vaccination. We recently showed that viable B. burgdorferi
sensu stricto 50772 could also be used to detect highly specific (>95%) borreliacidal antibodies without decreasing sensitivity (72%) by using a flow cytometric borreliacidal antibody test (4
). In this method, viable B. burgdorferi
sensu stricto 50772 is incubated with sera from patients with early Lyme borreliosis in the presence of complement. The enhanced specificity is due to the detection of only the antibodies that can specifically kill B. burgdorferi
. In contrast, bound cross-reactive antibodies are readily detected on non-viable B. burgdorferi
sensu stricto 50772 when exposed to conjugated anti-IgM or anti-IgG in the ELISA or IFA. Additional studies are being performed to determine whether this approach could eliminate the necessity of the two-tiered testing system.