Absence of Borrelia burgdorferi antibodies in the sera of Venezuelan patients with localized scleroderma (morphea). 

Fabiola Espinoza-León¹, Manzur Hassanhi-Hassanhi², Francisco Arocha-Sandoval¹ y Maricela Urbina-López¹. 

¹Departamento de Enfermedades Infecciosas y Tropicales y ²Departamento de Ciencias Fisiológicas. Facultad de Medicina, Universidad del Zulia. Maracaibo, Venezuela. E-mail: fabiolaespinoza@yahoo.com. 

Abstract. A possible association of Borrelia burgdorferi (Bb) with localized scleroderma (LS) has been postulated. However, the published data do not provide unequivocal results. Previous serologic analysis in LS patients in South American countries yielded positive results. The present study in our laboratory looked for evidence of Bb in LS patients in Venezuela, by antibacterial antibodies detection using the two-tiered approach as recommended by the Center for Disease Control and Prevention of Atlanta (United States). Twenty one serum samples from LS patients and twenty one samples from healthy individuals were analyzed. Four serum samples were ELISA positive: three from patients with LS and one from a healthy control. All ELISA positive samples were negative by IgG Western Blot. Our data do not support an association of Bb infection and the sclerotic lesions of LS; but do not rule out the possibility of a relationship between LS and an unknown geno-specie of Bb sensu lato complex, a different Borrelia specie or a different spirochetal organism, as the etiological agents of the skin lesions in the area. 

Key word: Borrelia burgdorferi, Lyme Borreliosis, localized scleroderma (morphea), Venezuela. 

Ausencia de anticuerpos contra Borrelia burgdorferi en suero de pacientes venezolanos con esclerodermia localizada (morfea).

Resumen. En los últimos años se ha postulado una posible asociación entre Borrelia burgdorferi (Bb) y esclerodermia localizada (morfea). Sin embargo, los datos publicados no han proporcionado pruebas inequívocas de tal asociación. En Suramérica se han realizado estudios serológicos que han evidenciado resultados positivos. El objetivo de este estudio fue buscar evidencia de la infección por Bb en pacientes venezolanos con morfea, mediante la detección de anticuerpos antibacterianos, usando el protocolo de dos pasos recomendado por el Center for Disease Control and Prevention de Atlanta (Estados Unidos). Veintiún muestras de suero de pacientes con morfea y veintiún muestras de sujetos sanos (control) fueron analizadas. Cuatro muestras resultaron ELISA positivas: tres correspondieron a pacientes con morfea y una, a un sujeto control. Todas las muestras ELISA positivas fueron negativas en la prueba IgG Western Blot. Los resultados obtenidos no soportan una asociación entre la infección por Bb y las lesiones escleróticas de morfea, pero tampoco descartan la posibilidad de una relación entre morfea y una genoespecie desconocida del complejo Bb sensu lato, entre morfea y una especie diferente de Borrelia o entre morfea y otra espiroqueta, como agente etiológico de la lesión en la localidad. 

Palabras clave: Borrelia burgdorferi, Borreliosis de Lyme, esclerodermia localizada (morfea), Venezuela. 

Received: 09-11-2005. Accepted: 23-02-2006. 

INTRODUCTION 

Localized scleroderma (LS), also known as morphea, is a disorder characterized by thickening and induration of the skin and subcutaneous tissues due to excessive collagen deposition (1). The disease has an incidence rate of 27 new cases per 1 million population per year. The actual incidence may be higher because many cases may not come to medical attention due to the benign nature of the disease (2). 

The etiology of morphea is still unknown. However, a possible association of Borrelia burgdorferi (Bb) with LS has been postulated. But the published data do not provide unequivocal results (3). 

In South America, antibodies against Bb have been detected in LS patients in Venezuela (Zulia State) and Colombia (4, 5). In the study conducted in Venezuelan patients, enzyme-linked immunosorbent assay (ELISA) positive results were not confirmed by immunoblot. 

To improve the accuracy of testing for antibodies to Bb, a two-tiered approach testing is currently recommended by the Center for Disease Control and Prevention (CDC) of Atlanta (United States). The two-steps protocol relies on a sensitive screening test (e.g. an ELISA), which is followed by specific immunoblotting assay of all samples with equivocal and positive screening test results (6). 

Considering that the detection of antibodies against Bb is a diagnostic method frequently used to analyze the association between LS and Bb (7), the aim of this study was to evaluate the presence of antibacterial antibodies in the sera of LS patients in Zulia State of Venezuela, using the two-tiered approach for antibodies detection against Bb as recommended by the CDC. 

MATERIALS AND METHODS 

Between July, 2002 and August, 2003, twenty one serum samples from LS patients in Zulia State of Venezuela were collected. The mean age of the patients was 21.7 years (range, 6-65 years). The clinical diagnosis of LS was confirmed by histological criteria. The onset of the clinical lesions was between 2 months and 7 years before sera samples were drawn. One of the patients remembered a tick bite. In addition, sera from 21 individuals (mean age, 30.5 years; range 17-61 years) without any symptoms of Lyme Borreliosis and who did not have morphea were taken as the control group. This study was approved by an ethics committee and the patients gave their informed consent. 

All samples were analyzed by ELISA using the C6 Lyme ELISA Kit, (Inmunetics, Inc. Cambridge, MA). The antigen used in this kit is a synthetic peptide, C6 peptide, derived from the VlsE protein of Bb. This peptide sequence is conserved and equally antigenic in humans infected with Bb sensu stricto or the European genospecies (B. garinni, B. afzelii). This assay detects total antibodies (IgM/IgG) and exhibits a sensitivity and specificity of 97%. Results were interpreted following the recommendations of the manufacturer. 

ELISA positive samples were analyzed by IgG Western Blot (WB) using QualiCode Lyme disease WB Kit, (Inmunetics, Inc. Cambridge, MA), which is elaborated with a low passage Bb B31 strain. This assay exhibits a sensitivity of 83% and a specificity of 97%. Results were interpreted following the recommendations of the manufacturer. 

RESULTS 

Four serum samples were ELISA positive: three of them were from patients with LS. The fourth positive sample corresponded to a healthy control. All ELISA positive samples were negative by IgG WB (Table I). 

TABLE I

IgG WB RESULTS OF STUDIED SAMPLES   

* Diagnostic Bands.    HC= Healthy control.    LS= Localized Scleroderma.    NC = Negative Control.    PC= Positive Control. 

DISCUSSION 

Since the relationship of Bb with morphea was first suggested by Aberer et al. (8) many investigations have been conducted to establish a possible association between the spirochetal infection and the skin lesion. Some studies, mostly European, using different techniques such as immunohistochemistry (9), culture (10-12), serology (10, 13-15) and polymerase chain reaction (16, 17) have demonstrated an association. On the contrary, North American studies have failed to do so (18-20). The association of the different clinical manifestations of Lyme Borreliosis and each genospecies of Bb (21) could partially explain these controversial results. Balmetti and Pifferetti (21), reported an association between cutaneous symptoms (e.g. erythema chronicum migrans and acrodermatitis chronica atrophicans) and the Bb genospecie, B. afzelii. Fujiwara et al. (20) detected B. afzelii and B. garinii (European genospecies), but not Bb sensu stricto, in skin biopsies of German and Japanese patients with morphea, using polymerase chain reaction assay. It is noteworthy to point out that the predominant strain in North America, Bb sensu stricto, has never been related with late dermatologic manifestations of Lyme Borreliosis (22). 

Although any of the Bb genospecies has been isolated in South America, positive serologic tests have been described in Venezuelan and Colombian patients with dermatologic manifestations associated with Lyme Borreliosis (4, 7, 23). Arocha et al. (4) detected antibodies against Bb in 6 of 14 Venezuelan patients suffering LS using ELISA. Sánchez et al. (23) reported a case of a young infant from Táchira State (Venezuela) with erythema chronicum migrans and antibodies against Bb. Few cases of erythema chronicum migrans have been detected in Zulia State of Venezuela (data not published), but an association between this condition and Bb infection has not been demonstrated yet. In Colombia, Palacios et al. reported IgG WB positive samples in 2 of 9 patients with morphea (5). 

Burkot et al. (24), reported that the interpretation of positive serology results in tropical countries, where neither Bb genospecies nor competent vectors have been isolated, should be done with caution, because of the possibility of cross-reactivity. Elevated immunoglobulin levels and cross-reactive antibodies against other pathogens (Treponema pallidum, Borrelia recurrentis, Helycobacter pylori, Ehrlichia, Babesia, Epstein-Barr virus) and diseases like systemic lupus erythematosus are likely sources of false reactivity in ELISAs of residents of tropical regions. The ability of WB to detect antibodies to individual proteins, including some specific of Bb, reduces the number of false positive test results obtained with ELISA (25). 

According to CDC criteria a negative result on the WB or ELISA indicates there is no serologic evidence of infection by Bb at the time the samples were drawn (6). Based on these criteria and considering the IgG WB assay negative results obtained in this study coupled with the lack of immunoblot confirmation of the ELISA positive results previously detected in the state, we considered the possibility that results reported by Arocha et al. were false-positives, due to cross-reactivity. 

Our data do not support an association of Bb infection and the sclerotic lesions of LS, but do not rule out the possibility of a relationship between LS and an unknown genospecie of Bb sensu lato complex, different Borrelia specie or a different spirochetal organism, as the etiological agents of the skin lesions. A study conducted in Colombia reported a partial shared reactivity of sera from patients with syphilis and LS, providing evidence for a possible association of sclerotic lesions with a spirochetal organism (7). 

Our long-term goals include new studies using other methodologies such as polymerase chain reaction in skin biopsies of LS patients to further investigate the association of LS and Bb infection in the area and a survey of competent vectors in Zulia State. 

ACKNOWLEDGEMENTS 

We thank Mrs. Teresa Roman for her excellent laboratory skills and Dr. James Kumi-Diaka for his critically review of the manuscript. 

REFERENCES 

1. Sehgal VN, Srivastava G, Aggarwal AK, Behl PN, Choudhary M, Bajaj P. Localized scleroderma/morphea. Int J Dermatol 2002; 41:467-475.         [ Links ]

2. Peterson LS, Nelson AM, Su WP, Mason T, O’Fallon WM, Gabriel SE. The epidemiology of morphea (localized scleroderma) in Olmsted County 1960-1993. J Rheumatol 1997; 24:73-80.         [ Links ]

3. Weide B, Walz T, Garbe C. Is morphoea caused by Borrelia burgdorferi? A review. Br J Dermatol 2000; 142:636-644.         [ Links ]

4. Arocha F, Amesty A, Urbina M, Durango A, Vargas H. Detección de anticuerpos contra Borrelia burgdorferi en una muestra poblacional del Estado Zulia. Invest Clin 1994; 35:91-104.         [ Links ]

5. Palacios R, Osorio L, Giraldo L, Torres A, Philipp M, Ochoa M. Positive IgG Western Blot for Borrelia burgdorferi in Colombia. Mem Inst Oswaldo Cruz 1999; 94:499-503.         [ Links ]

6. Center for Disease and Control Prevention. Recommendations for Test Performance and Interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. Morb Mort Wkly Rep 1995; 44:590-591.         [ Links ]

7. Palacios R, Torres A, Trujillo R. IgG antibody reactivity to Borrelia burgdorferi sensu stricto antigens in patients with morphea in Colombia. Int J Dermatol 2003; 42:882-886.         [ Links ]

8. Aberer E, Neumann R, Stanek G. Is localized scleroderma a borrelia infection?. Lancet 1985; 2:278.         [ Links ]

9. Aberer E, Stanek G. Histological evidence for spirochetal origin of morphea and lichen sclerosus et atrophicans. Am J Dermatopathol 1987; 9:374-379.         [ Links ]

10. Aberer E, Stanek G, Ertl M, Neumann R. Evidence for spirochetal origin of circumscribed scleroderma (morphea). Acta Derma Venereol 1987; 67:225-231.         [ Links ]

11. Weber K, Preac-Mursic V, Reimers CD. Spirochetes isolated from two patients with morphea. Infection 1988; 16:25-26.         [ Links ]

12. Breier FH, Aberer E, Stanek G, Khanakaha G, Schlick A, Tappeiner G. Isolation of Borrelia afzelii from circumscribed scleroderma. Br J Dermatol 1999; 140:925-930.         [ Links ]

13. Aberer E, Klade H, Stanek G, Gebhart W. Borrelia burgdorferi and different types of morphea. Dermatologica 1991; 82:145-154.         [ Links ]

14. Nakashima T, Maeda M, Hayashi T, Kitamura K. A case of generalized morphea with a high titer of anti-Borrelia burgdorferi antibodies. J Dermatol 1999; 26:821-824.         [ Links ]

15. Wojas-Pelc A, Wielowieyska-Szybinska D, Kieltyka A. Presence of the antinuclear antibodies and antibodies to Borrelia burgdorferi among patients with morphea en plaque, deep linear scleroderma and atrophoderma Pasini-Pierini. Przegl Lek 2002; 59:898-902.         [ Links ]

16. Schempp C, Bocklage H, Lange R, Kolmel H, Orfanos C, Gollnick H. Further evidence for Borrelia burgdorferi infection in morphea and lichen sclerosus et atrophicus confirmed by DNA amplification. J Invest Dermatol 1993; 100:717-720.         [ Links ]

17. Ozkan S, Atabey N, Fetil E, Erkizan V, Gunes A. Evidence for Borrelia burgdorferi in morphea and lichen sclerosus. Int J Dermatol 2000; 39:278- 283.         [ Links ]

18. Dillon W, Saed G, Fivenson D. Borrelia burgdorferi DNA is undetectable by PCR in skin lesions of morphea, schleroderma or lichen sclerosus et atrophicus of patients from North America. J Am Acad Dermatol 1995; 33:617-620.         [ Links ]

19. De Vito J, Merogi A, Vo T, Boh E, Fung H, Freeman SM, Cockerell C, Stewart K, Marrogi AJ. Role of Borrelia burgdorferi in the pathogenesis of morphea/scleroderma and lichen sclerosus et atrophicus: a PCR study of thirty-five cases. J Cutan Pathol 1996; 23:350-358.          [ Links ]

20. Fujiwara H, Fujiwara K, Hashimoto K, Mehregan AH, Schaumburg-Lever G, Lange R, Schempp C, Gollnick H. Detection of Borrelia burgdorferi DNA (B. garinii or B. afzelii) in morphea and lichen sclerosus et atrophicus tissues of German and Japanese but not of US patients. Arch Dermatol 1997; 133:41-44.         [ Links ]

21. Balmetti T, Pifferetti J. Association between different clinical manifestations of Lyme disease and different species of Borrelia burgdorferi sensu lato. Res Microbiol 1995; 146:329-340.         [ Links ]

22. Schmidt BL. PCR in laboratory diagnosis of human Borrelia burgdorferi infections. Clin Microbiol Rev 1997; 10:185-201.         [ Links ]

23. Sánchez B, Álvarez C, Morales Z, Cherubini B. Enfermedad de Lyme: reporte de un caso. Rev. Col. med. estado Táchira 1998: 7:41-42.         [ Links ]

24. Burkot T, Schriefer M, Larsen S. Cross-reactivity to Borrelia burdorferi proteins in serum samples from residents of a tropical country nonendemic for Lyme Disease. J Infect Dis 1997; 175:466-469.         [ Links ]

25. Brown S, Hansen S, Langone J. Role of serology in the diagnosis of Lyme disease. JAMA 1999; 282:62-66.         [ Links ]

Autor de correspondencia: Fabiola Espinoza-León. Departamento de Enfermedades Infecciosas y Tropicales, Facultad de Medicina, Universidad del Zulia. Maracaibo, Venezuela. Teléfonos: 58-2617929652 58-2617597229 Correo electrónico: fabiolaespinoza@yahoo.com.