A novel lymphocyte transformation test (LTT-MELISA®) for Lyme borreliosis

Abstract

Diagnosis of active Lyme borreliosis (LB) remains a challenge in clinically ambiguous, serologically indeterminant, and polymerase chain reaction-negative patients. Lymphocyte transformation tests (LTTs) have been applied to detect specific cellular immune reactivity, but their clinical application has been severely hampered by the poorly defined Borrelia antigens and nonstandardized LTT formats used. In this study, we describe the development and clinical relevance of a novel LTT using a validated format (MELISA®) together with well-defined recombinant Borrelia-specific antigens. From an initial screening of 244 patients with suspected Borrelia infection or disease, 4 informative recombinant antigens were selected: OspC (Borrelia afzelii), p41-1 (Borrelia garinii), p41-2 (B. afzelii), and p100 (B. afzelii). Thereafter, 30 seronegative healthy controls were tested in LTT-MELISA® to determine specificity, 68 patients were tested in parallel to determine reproducibility, and 54 lymphocyte-reactive symptomatic patients were tested before and after antibiotic therapy to assess clinical relevance. Most (86.2%) of the 36.9% (90/244) LTT-MELISA® positive patients were seropositive and showed symptoms of active LB. Specificity was 96.7% and reproducibility 92.6%. After therapy, most patients (90.7%) showed negative or markedly reduced lymphocyte reactivity correlating with clinical improvement. This novel LTT-MELISA® assay appears to correlate with active LB and may have diagnostic relevance in confirming LB in clinically and serologically ambiguous cases.

Introduction

Lyme borreliosis (LB) is the most frequent vector-borne disease in Europe and the United States, and its prevalence appears to be rising (Centers for Disease Control and Prevention, 2004). The disease is transmitted by ticks harboring the infectious agent Borrelia burgdorferi sensu latu. Although all 3 human pathogenic species B. burgdorferi sensu stricto, B. afzelii, and Borrelia garinii cause LB in Europe, B. burgdorferi sensu stricto appears to be the sole etiologic agent in the United States (reviewed in Stanek and Strle, 2003, Steere et al., 2004).

A multisystem disorder, LB produces a wide range of clinical manifestations. Only erythema migrans (EM), a transient reddening of the skin circumventing the tick bite site and developing within days to weeks after infection, may be pathognomonic, but at the same time, it occurs in only 60% of LB patients. All other extracutaneous symptoms are characteristic of a variety of diseases, rendering clinical diagnosis alone, except in the case of EM, as problematic (Stanek and Strle, 2003, Steere et al., 2004).

Routine laboratory testing is based on detecting the humoral immune response to Borrelia-specific antigens in a 2-tiered format: screening with an enzyme-linked immunoassay (ELISA) followed by confirmation of positive or borderline results in a more specific immunoblot or Western blot. However, although serologic analyses can confirm infection, they do not prove the existence of the disease. In addition, serologic results are frequently negative in early Lyme disease, antibodies may persist for several years despite successful therapy, and interlaboratory serologic results may be discordant (Robertson et al., 2000, Wilske, 2003, Aguero-Rosenfeld et al., 2005). Microbial detection by culture or polymerase chain reaction (PCR) is highly specific but has a limited sensitivity (Dumler, 2001). Therefore, although infection with B. burgdorferi can often be determined, diagnosis of the disease itself remains challenging. At the same time, early therapeutic intervention appears to play a crucial role in preventing disease progression (Dattwyler et al., 1988).

During the 1990s, several groups attempted to improve the diagnosis of LB by examining the specific cellular immune reactivity to B. burgdorferi using various forms of the lymphocyte transformation test (LTT) (Table 1) (Dattwyler et al., 1988, Dressler et al., 1991, Krause et al., 1991, Yoshinari et al., 1991, Zoschke et al., 1991, Krause et al., 1992, Schempp et al., 1993, Roessner et al., 1994, Breier et al., 1995, Huppertz et al., 1996, Rutkowski et al., 1997). Clinical application of LTT for Lyme disease, however, proved limited because of generally poor sensitivity, specificity, and reproducibility, and its use has since been discouraged (Wilske, 2003). Likely causes of these poor results include the use of 1) nonvalidated nonstandardized LTT formats with low lymphocyte concentrations (<250 000 cells per test), and 2) poorly defined antigenically heterogeneous “lysates” from whole B. burgdorferi or culture supernatants as antigens.

In the present study, we describe a novel LTT that incorporates 2 improvements to obviate the abovementioned problems. First, a standardized LTT format using a higher cell concentration (1 × 10⁶ cells per test), memory lymphocyte immunostimulation assay (MELISA®), was applied (Stejskal et al., 1994). Its technical validity and clinical relevance for detecting cellular immune reactivity to other antigens has been extensively documented (Stejskal et al., 1986, Stejskal et al., 1990, Stejskal et al., 1996, Stejskal et al., 1999, Tibbling et al., 1995, Stejskal, 1997, Sterzl et al., 1999, Regland et al., 2001, Valentine-Thon and Schiwara, 2003, Prochazkova et al., 2004, Kakuschke et al., 2005). Second, only well-characterized recombinant Borrelia-specific antigens were used (Jauris-Heipke et al., 1993a, Jauris-Heipke et al., 1993b, Luft et al., 1993, Robinson et al., 1993, Wilske et al., 1993a, Wilske et al., 1993b, Wilske, 2003). The specificity, reproducibility, and clinical potential of this novel LTT-MELISA® for LB are presented.