Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

Difficulties in HAM/TSP diagnosis / Dificuldades no diagnóstico da HAM/TSP

The World Health Organization recommends the use of Osame's criterion (1990) for the diagnosis of HTLV-I-associated myelopathy (HAM/TSP). In 2006, a group of neurologists developed a Brazilian criterion that can diagnose HAM/TSP from its onset. OBJECTIVE: It was to test the agreement between both criteria. METHODS: The study included evaluation of clinical and laboratory findings of 35 patients. The ELISA, Western blot and/or polymerase chain reaction was used to search for anti-HTLV-I antibodies. The analysis of agreement was based on the calculation of Kappa. RESULTS: Concordance of 100% (Kappa=1) occurred in cases of "defined" HAM/TSP, but not in patients with "probable" diagnosis. CONCLUSION: The Brazilian criteria was as effective as Osame's criteria for the diagnosis of "defined" HAM/TSP. However, both require more specific biological markers in cerebrospinal fluid for the laboratory diagnosis of probable cases.

A Organização Mundial da Saúde recomenda o uso do critério de Osame (1990) para o diagnóstico da mielopatia associada ao vírus HTLV-I (HAM/TSP). Em 2006, um grupo de neurologistas elaborou um critério brasileiro capaz de diagnosticar HAM/TSP desde suas manifestações iniciais. OBJETIVO: Foi testar a concordância entre ambos os critérios. MÉTODOS: O estudo incluiu a avaliação dos achados clínicos e laboratoriais de 35 pacientes. Os métodos de ELISA, Western blot e/ou reação em cadeia da polimerase foram utilizados para pesquisa de anticorpos anti-HTLV-I. A análise da concordância baseou-se no cálculo do índice Kappa. RESULTADOS: Concordância de 100% (índice Kappa=1) ocorreu nos casos de HAM/TSP "definida", mas não nos pacientes com o diagnóstico "provável". CONCLUSÃO: O critério brasileiro foi tão eficaz quanto o critério de Osame para o diagnóstico de HAM/TSP "definido". No entanto, ambos necessitam de marcadores biológicos mais específicos no líquido cefalorraquidiano para o diagnóstico laboratorial dos casos prováveis.