Interpretation criteria for standardized Western blots for three European species of Borrelia burgdorferi sensu lato

Abstract

Western blots (WBs; immunoblots) are a widely used tool for the serodiagnosis of Lyme borreliosis, but so far, no defined criteria for performance, analysis, and interpretation have been established in Europe. For the current study WBs were produced with strains PKa2 (Borrelia burgdorferi sensu stricto), PKo (Borrelia afzelii), and PBi (Borrelia garinii). To improve resolution we used gels of 17 cm in length. In a first step, 13 immunodominant proteins were identified with monoclonal antibodies. Then, the apparent molecular masses of all visually distinguishable bands were determined densitometrically. Approximately 40 bands of between 14 and 100 kDa were differentiated for each strain. From a study with 330 serum samples (from 189 patients with Lyme borreliosis and 141 controls), all observed bands were documented. To establish criteria for a positive WB result, the discriminating ability of a series of band combinations (interpretation rules) were evaluated separately for each strain (for immunoglobulin G [IgG] WB, >40 combinations; for IgM WB, >15 combinations). The following interpretation criteria resulting in specificities of greater than 96% were recommended: for IgG WB, at least one band of p83/100, p58, p56, OspC, p21, and p17a for PKa2; at least two bands of p83/100, p58, p43, p39, p30, OspC, p21, p17, and p14 for PKo; and at least one band of p83/100, p39, OspC, p21, and p17b for PBi; for IgM WB, at least one band of p39, OspC, and p17a or a strong p41 band for PKa2; at least one band of p39, OspC, and p17 or a strong p41 band for PKo; and at least one band of p39 and OspC or a strong p41 band for PBi. The overall sensitivity was the highest for PKo WB, followed by PBi and PKa2 WB, in decreasing order. Standardization of WB assays is necessary for comparison of results from different laboratories.

IgG antibody reactivity to Borrelia burgdorferi sensu stricto antigens in patients with morphea in Colombia

“…To distinguish between antibodies against B. burgdorferi induced by the putative etiologic agent and cross-reactivity with other bacteria, the frequency of antibodies to individual antigenic bands in the sera of these patients was compared with that of patients with syphilis and healthy individuals as negative controls. 6,7,13 Antigenic band patterns were analyzed to determine the pattern which could discriminate between patients with morphea/ LSA and controls. The resulting antigenic band pattern was then used as a criterion to determine possible exposure to a putative spirochete causing morphea/LSA.…”

“…Available commercial immunoblot kits prepared with this genospecies and immunoblots based on Borrelia burgdorferi sensu stricto can detect other related spirochetes, such as B. afzelii and B. garinii . 5,6 The criteria of Dressler et al . 7 are used in North America for the interpretation of immunoglobulin G (IgG) immunoblots.…”

“…These criteria are not as useful for other countries, however, where genospecies of B. burgdorferi sensu lato different from B. burgdorferi sensu stricto exist. 5,6,8 Therefore, studies to identify other criteria for the interpretation of immunoblots in these countries have been conducted. 5,6 In Latin America, where there are no records of the isolation of any genospecies of B. burgdorferi , the detection of antibodies against B. burgdorferi associated with morphea has been reported in Venezuela 9 and Colombia.…”

“…5,6,8 Therefore, studies to identify other criteria for the interpretation of immunoblots in these countries have been conducted. 5,6 In Latin America, where there are no records of the isolation of any genospecies of B. burgdorferi , the detection of antibodies against B. burgdorferi associated with morphea has been reported in Venezuela 9 and Colombia. 10,11 These findings are held in low esteem because of the general belief that Lyme borreliosis does not exist in tropical countries and that positive serologies, including immunoblots, are due to cross-reactivity.…”

Microbiological and serological diagnosis of Lyme borreliosis

“…strain PKo) (Wilske et al, 2000). The immunoblot criteria recommended by the Centers for Disease Control (CDC) that are used in the USA are not applicable for European patients (Hauser et al, 1997(Hauser et al, , 1998Robertson et al, 2000). Dressler et al (1994) have shown, in an immunoblot study, that the immune response of European patients is restricted to a narrower spectrum of Borrelia proteins, compared with that shown by American patients.…”