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.

Diagnóstico laboratorial da mielopatia associada ao HTLV-I: métodos para análise do líquido cefalorraquidiano / Laboratorial diagnosis of HTLV-I associated myelopathy: methods for the cerebrospinal fluid analysis

O vírus linfotrópico de células T humanas do tipo I (HTLV-I) pode causar uma doença neurológica inflamatória, crônica e incapacitante, que acomete a medula espinhal, denominada mielopatia associada ao HTLV-I/paraparesia espástica tropical (PET/MAH). A verificação de anticorpos da classe G (IgG) anti-HTLV-I no soro e no líquido cefalorraquidiano (LCR) representa importante parâmetro para o diagnóstico laboratorial da PET/MAH. OBJETIVO: Avaliação crítica dos métodos utilizados para verificação da presença e da produção intratecal de anticorpos totais e anti-HTLV-I no LCR para o diagnóstico de PET/MAH. MÉTODO: Realizou-se uma revisão sistemática de artigos da literatura médica, usando-se palavras-chave da língua inglesa como cerebrospinal fluid, intrathecal synthesis of antibodies, HTLV-I, HAM/TSP. As bases de dados utilizadas incluíram Pubmed, Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs), MEDlars onLINE (Medline) e Cochrane Library. RESULTADO: Foram selecionados 14 artigos: cinco relacionados com a presença do anticorpo IgG específico no LCR; nove sobre síntese intratecal de anticorpos totais (IgG ou IgG/IgA/IgM) e específicos anti-HTLV-I (IgG ou IgM). DISCUSSÃO: O estudo isolado da presença de anticorpo IgG anti-HTLV-I no LCR não discrimina a fração produzida no sistema nervoso central (SNC), possui baixa especificidade (40 por cento) para o diagnóstico de PET/MAH. A demonstração da síntese intratecal de anticorpos IgG anti-HTLV-I possui maior relevância por suas elevadas especificidade (89 por cento) e sensibilidade (83 por cento). Entre os métodos para a avaliação da síntese intratecal de anticorpo específico, destaca-se o índice de IgG anti-HTLV-I, segundo Reiber e Felgenhauer(18), o qual se baseia no teste do ensaio imunossorvente ligado à enzima (ELISA), com análise simultânea do LCR e do soro. Outros estudos utilizam pequenas amostragens e não demonstram sensibilidade e especificidade no teste do LCR...

The human T-cell lymphotropic virus type I (HTLV-I) may cause HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP), an incapacitating chronic inflammatory disease of the spinal cord. The detection of IgG anti-HTLV-I antibodies in the serum and cerebrospinal fluid (CSF) has been an important parameter for the laboratorial diagnosis of HAM/TSP. OBJECTIVE: critical evaluation of the methods applied to detect the presence and intrathecal production of total antibodies and anti-HTLV-I in the CSF for the diagnosis of HAM/TSP. METHODS: We performed a systematic review of medical articles by using the key words: "cerebrospinal fluid, intrathecal synthesis of antibodies, HTLV-I associated myelopathy, HTLV-I, HAM/TSP". The used databases included: PubMed, Lilacs, Medline and Cochrane Library. RESULTS: A total of 14 articles were selected: five studies were related to the presence of specific IgG antibody in the CSF and nine studied the intrathecal synthesis of total antibodies (IgG or IgG/IgA/IgM) and specific anti-HTLV-I (IgG or IgM). DISCUSSION: The isolated study of the presence of IgG antibody anti-HTLV-I in the CSF does not show the fraction produced in the central nervous system, which represents low specificity (40 percent) for the diagnosis of HAM/TSP. The demonstration of the intrathecal synthesis of IgG anti-HTLV-I antibodies is more relevant due to its high specificity (89 percent) and sensibility (83 percent). According to Reiber & Felgenhauer (1987), the index IgG anti-HTLV-I, which is based on ELISA test with simultaneous CSF and serum analysis, stands out from the other methods applied to evaluate the intrathecal synthesis of specific antibody. Other studies use small samples and do not demonstrate the sensibility and specificity of the test in the CSF. Only one study shows statistical analysis. CONCLUSION: The immunological diagnosis of the CSF in HAM/TSP requires the standardization of methods, which should be based...