Falha na implantação de um novo algoritmo de testes laboratoriais para o diagnóstico de infecção por HTLV-1 e HTLV-2 em população de risco / Lack in introducing a new algorithm of laboratorial tests for detecting HTLV-1 and HTLV-2 infections in at-risk population

Em vista dos problemas detectados no diagnóstico de infecção pelos vírus linfotrópicos de células T humanas dos tipos 1 e -2 (HTLV-1 e HTLV-2) em casuística encaminhada ao Instituto Adolfo Lutz de São Paulo, foi proposto um novo algoritmo de testes laboratoriais que utiliza duas amostras de sangue seqüenciais. Na primeira o sangue é coletado em tubo seco e feita triagem sorológica com dois ensaios imunoenzimáticos (EIAs). Na segunda, o sangue é coletado em tubo contendo o anticoagulante ácido etilenodiamino tetra acético (EDTA) para a repetição dos EIAs e para os testes confirmatórios de Western blot (WB) e reação em cadeia da polimerase (PCR). Os resultados obtidos com 313 amostras de sangue mostraram ineficiência do algoritmo, pois nos casos EIA reagentes, apenas 25% tiveram uma segunda amostra de sangue coletada e destas, apenas três em EDTA. Portanto, não foi possível comparar o desempenho da PCR em relação ao WB. Um algoritmo simples, de coleta única de sangue em tubo contendo EDTA foi proposto e vem sendo utilizado para a triagem e para os testes confirmatórios.

Confirming the presence of HTLV-1 infection and the absence of HTLV-2 in blood donors from Arequipa, Peru.

Epidemiological studies conducted in Peru disclosed HTLV-1 to be prevalent in different ethnic groups, and found HTLV-2 in some Amazonian Indians and in men who have sex with men. No data concerning HTLV-1/2 infection in blood donors from Arequipa, a highlands region in southern Peru, is available. We searched for the presence of HTLV-1 and HTLV-2 antibodies in 2,732 serum samples obtained from blood donors from this geographic area. HTLV-1/2-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA) and were confirmed by Western blot (WB). Reactive sera had their blood bags discarded from donation, and the demographic characteristics of the donors were analyzed. Thirty-five sera (1.2%) were HTLV seroreactive by ELISA, and 25 were confirmed HTLV-1-positive by WB. One serum disclosed HTLV-positivity, and the remaining nine serum samples showed indeterminate results by WB; three of which had an HTLV-1 indeterminate Gag profile. The median age of HTLV-positive individuals was 34.6 years; 27 were male and eight were female. All individuals were from southern Peru: 27 from Arequipa, five from Puno, and three from Cuzco. HTLV co-positivity with hepatitis B (five sera) and syphilis (one serum) were detected. Previous transfusion and tattooing were observed in two and one individuals, respectively. No serum was positive for HTLV/HIV co-infection. This study confirmed, for the first time, HTLV-1 infection and the absence of HTLV-2 infection in blood donors from Arequipa, Peru and suggests vertical transmission as the major route of HTLV-1 transmission and acquisition in this geographic region.

Confirming the presence of HTLV-1 infection and the absence of HTLV-2 in blood donors from Arequipa, Peru / Confirmação da presença de infecção por HTLV-1 e ausência de infecção por HTLV-2 em doadores de sangue de Arequipa, Peru

Epidemiological studies conducted in Peru disclosed HTLV-1 to be prevalent in different ethnic groups, and found HTLV-2 in some Amazonian Indians and in men who have sex with men. No data concerning HTLV-1/2 infection in blood donors from Arequipa, a highlands region in southern Peru, is available. We searched for the presence of HTLV-1 and HTLV-2 antibodies in 2,732 serum samples obtained from blood donors from this geographic area. HTLV-1/2-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA) and were confirmed by Western blot (WB). Reactive sera had their blood bags discarded from donation, and the demographic characteristics of the donors were analyzed. Thirty-five sera (1.2 percent) were HTLV seroreactive by ELISA, and 25 were confirmed HTLV-1-positive by WB. One serum disclosed HTLV-positivity, and the remaining nine serum samples showed indeterminate results by WB; three of which had an HTLV-1 indeterminate Gag profile. The median age of HTLV-positive individuals was 34.6 years; 27 were male and eight were female. All individuals were from southern Peru: 27 from Arequipa, five from Puno, and three from Cuzco. HTLV co-positivity with hepatitis B (five sera) and syphilis (one serum) were detected. Previous transfusion and tattooing were observed in two and one individuals, respectively. No serum was positive for HTLV/HIV co-infection. This study confirmed, for the first time, HTLV-1 infection and the absence of HTLV-2 infection in blood donors from Arequipa, Peru and suggests vertical transmission as the major route of HTLV-1 transmission and acquisition in this geographic region.

Estudos epidemiológicos conduzidos no Peru apontam a infecção por HTLV-1 como prevalente em diferentes grupos étnicos e por HTLV-2 restrita a alguns índios da região Amazônica e a homens que fazem sexo com homens. Não existem dados sobre a infecção por HTLV-1/2 em doadores de sangue de Arequipa, região montanhosa do sul do Peru. Portanto, o presente estudo pesquisou anticorpos anti-HTLV-1 e HTLV-2 em 2.732 doadores de sangue desta região geográfica. Foram utilizados na triagem sorológica os testes imunoenzimáticos (ELISA) e para confirmação dos resultados o Western Blot (WB). Soros reagentes no ELISA tiveram suas bolsas de sangue descartadas. Os resultados obtidos foram analisados de acordo com características demográficas dos indivíduos. Trinta e cinco soros (1,2 por cento) resultaram HTLV-1/2 reagentes no ELISA, 25 confirmaram infecção por HTLV-1 no WB. Um soro resultou HTLV positivo e os nove soros restantes resultaram em padrão indeterminado no WB: três com perfil HTLV-1 Gag indeterminado. A média de idade dos indivíduos HTLV positivos foi de 34,6 anos; 27 do gênero masculino e oito do gênero feminino. Todos eram da região sul do país: 27 de Arequipa, cinco de Puno e três de Cuzco. Foi detectada co-positividade HTLV com hepatite B (cinco soros) e sífilis (um soro). Nenhum soro resultou positivo para a co-infecção HIV/HTLV. Havia dois indivíduos com transfusão prévia e um com tatuagem. Este trabalho confirma pela primeira vez infecção por HTLV-1 e ausência de infecção por HTLV-2 em doadores de sangue de Arequipa, sul do Peru e sugere que a transmissão vertical seja a principal via de transmissão/aquisição de HTLV-1 nesta região geográfica.

Algoritmo de testes sorológicos de triagem para infecção por HTLV-1/2 usado no Instituto Adolfo Lutz / Algorithm of HTLV-1/2 serological screening tests employed by Instituto Adolfo Lutz

O Ministério da Saúde recomenda para o diagnóstico de infecção por HTLV-1/2 a utilização de um ensaio imunoenzimático (EIA) que contenha antígenos específicos do HTLV-1 e HTLV-2 na triagem sorológica e, nos soros reagentes, a repetição do mesmo teste em duplicata. O presente artigo tem como objetivo apresentar os resultados obtidos com a rotina diagnóstica de infecção por HTLV-1/2 e o algoritmo de testes de triagem utilizado pelo Instituto Adolfo Lutz Central, que difere do recomendado pelo Ministério. Amostras de soro encaminhadas por ambulatórios de especialidades do SUS e pelos Centros de Referência e Treinamento em AIDS de São Paulo, no período de dezembro de 1998 a março de 2006, foram analisadas quanto à presença de anticorpos anti-HTLV-1/2, usando kits de reagentes EIAs disponíveis no mercado. Os resultados obtidos são apresentados como valores de sensibilidade, especificidade e eficiência dos EIAs de primeira, segunda e terceira gerações em relação ao teste confirmatório de Western Blot, destacando os resultados falso-negativos. Nenhum kit de reagentes EIA, isoladamente, foi capaz de detectar todos os soros verdadeiramente positivos quanto à presença de anticorpos anti-HTLV-1/2. Houve necessidade da utilização de dois EIAs de composição antigênica e formatos diferentes para a triagem sorológica, com melhor desempenho dos EIAs de segunda geração. Os resultados obtidos confirmam a necessidade da utilização de dois kits de reagentes EIAs na triagem sorológica de infecção por HTLV-1/2 em laboratórios de diagnóstico, diferentemente do preconizado pelo Ministério da Saúde.

Performances of HTLV serological tests in diagnosing HTLV infection in high-risk population of São Paulo, Brazil.

Testing problems in diagnosing human T-lymphotropic virus (HTLV) infection, mostly HTLV-II, have been documented in HIV/AIDS patients. Since December 1998, the Immunology Department of Instituto Adolfo Lutz (IAL) offers HTLV-I/II serology to Public Health Units that attend HTLV high-risk individuals. Two thousand, three hundred and twelve serum samples: 1,393 from AIDS Reference Centers (Group I), and 919 from HTLV out-patient clinics (Group II) were sent to IAL for HTLV-I/II antibodies detection. The majority of them were screened by two enzyme immunoassays (EIAs), and confirmed by Western Blot (WB 2.4, Genelabs). Seven different EIA kits were employed during the period, and according to WB results, the best performance was obtained by EIAs that contain HTLV-I and HTLV-II viral lysates and rgp21 as antigens. Neither 1st and 2nd, nor 3rd generation EIA kits were 100% sensitive in detecting truly HTLV-I/II reactive samples. HTLV-I and HTLV-II prevalence rates of 3.3% and 2.5% were detected in Group I, and of 9.6% and 3.6% in Group II, respectively. High percentages of HTLV-seroindeterminate WB sera were detected in both Groups. The algorithm testing to be employed in HTLV high-risk population from São Paulo, Brazil, needs the use of two EIA kits of different formats and compounds as screening, and because of high seroindeterminate WB, may be another confirmatory assay.

Performances of HTLV serological tests in diagnosing HTLV infection in high-risk population of São Paulo, Brazil

Testing problems in diagnosing human T-lymphotropic virus (HTLV) infection, mostly HTLV-II, have been documented in HIV/AIDS patients. Since December 1998, the Immunology Department of Instituto Adolfo Lutz (IAL) offers HTLV-I/II serology to Public Health Units that attend HTLV high-risk individuals. Two thousand, three hundred and twelve serum samples: 1,393 from AIDS Reference Centers (Group I), and 919 from HTLV out-patient clinics (Group II) were sent to IAL for HTLV-I/II antibodies detection. The majority of them were screened by two enzyme immunoassays (EIAs), and confirmed by Western Blot (WB 2.4, Genelabs). Seven different EIA kits were employed during the period, and according to WB results, the best performance was obtained by EIAs that contain HTLV-I and HTLV-II viral lysates and rgp21 as antigens. Neither 1st and 2nd, nor 3rd generation EIA kits were 100 percent sensitive in detecting truly HTLV-I/II reactive samples. HTLV-I and HTLV-II prevalence rates of 3.3 percent and 2.5 percent were detected in Group I, and of 9.6 percent and 3.6 percent in Group II, respectively. High percentages of HTLV-seroindeterminate WB sera were detected in both Groups. The algorithm testing to be employed in HTLV high-risk population from São Paulo, Brazil, needs the use of two EIA kits of different formats and compounds as screening, and because of high seroindeterminate WB, may be another confirmatory assay.

Problemas nos testes diagnósticos de infecção pelos vírus linfotrópicos de células T humanas (HTLV), principalmente HTLV-II, têm sido observados em pacientes com HIV/Aids. Desde Dezembro de 1998, a Seção de Imunologia do Instituto Adolfo Lutz (IAL) oferece a sorologia para HTLV-I/II para Serviços de Saúde Pública que atendem populações consideradas de risco para esta infecção. Duas mil trezentas e doze amostras de soro: 1.393 de Centros de Referência em Aids (Grupo I) e 919 de Clínicas de Especialidade em HTLV (Grupo II) foram encaminhadas para o IAL para a pesquisa de anticorpos anti-HTLV-I/II. A maioria delas foram testadas por dois ensaios imunoenzimáticos (EIAs) e confirmadas por Western Blot (WB 2.4, Genelabs). Sete kits diferentes de EIAs foram empregados durante o período e de acordo com os resultados do WB a melhor performance foi obtida com os EIAs que continham lisado viral dos HTLV-I e -II e a rgp21 como antígenos. Nenhum kit de EIA de 1ª, 2ª ou 3ª geração foi 100 por cento sensível para detectar todas as amostras verdadeiramente HTLV-I/II reagentes. A prevalência de HTLV-I e HTLV-II, respectivamente, foi de 3,3 por cento e 2,5 por cento no Grupo I e de 9,6 por cento e 3,6 por cento no Grupo II. Em ambos os Grupos, foram detectadas altas percentagens de soros com padrão indeterminado no WB. O algoritmo de testes sorológicos para ser usado em população de alto risco para HTLV de São Paulo, Brasil, necessita de dois kits EIAs de princípios e composição diferentes para a triagem sorológica e, pelo elevado número de WB indeterminado, talvez de um outro teste confirmatório.

Otimização e avaliação do ensaio de imunofluorescência indireta para pesquisa de anticorpos anti-herpesvírus humano 8 (HHV-8) usando sangue colhido em papel de filtro / Standardization of the indirect immunofluorescence assay to search for human herpesvirus 8 (HHV-8) antibodies using blood collected on filter paper

O presente trabalho objetivou a otimização e a avaliação da coleta de sangue em papel de filtro para ser usada em levantamentos soroepidemiológicos de infecção por herpes vírus humano 8 (HHV-8). Foram utilizados os ensaios de imunofluorescência indireta (IFI) LANA e Lítico para a pesquisa de anticorposanti-HHV-8 em amostras de sangue colhidas em membrana Schleicher Schuell de 28 pacientes com sarcomade Kaposi (SK)/AIDS e 10 indivíduos sadios sem SK, dos quais os soros haviam sido anteriormente usados na padronização destas técnicas. Lâminas contendo células da linhagem BCBL-1 estimuladas ou não com éster de forbol, e luatos de sangue e conjugado anti-imunoglobulina humana marcado com fluor esceína foram empregados nas reações sorológicas e, as reações e os critérios de positividade seguiram os previamente estabelecidos na Seção de Imunologia do Instituto Adolfo Lutz. Foi detectada fluorescência verde-amarelada na maioria das células nas diluições 1:50 e 1:100. Isto dificultou a leitura da IFI-Lítico, principalmente em diluições baixas, mas não interferiu na IFI-LANA cujo padrão pontilhado no núcleo coexistiu com a fluorescência de membrana. À medida que se seguiram as diluições, houve desaparecimento da inespecificidade e a leitura prosseguiu normalmente. Houve concordância de resultados positivos e negativos, com diferenças mínimas nos títulos de anticorpos. Os resultados obtidos viabilizam a coleta de sangue em papel para estudos epidemiológicos de infecção por HHV-8.

Seroprevalence of HHV-8 infection in the pediatric population of two university hospitals in Rio de Janeiro, Brazil.

BACKGROUND: The authors present a serologic survey of the prevalence of herpesvirus-8 (HHV-8) among 171 children from 0 to 12 years old in Rio de Janeiro, Brazil. MATERIALS AND METHODS: Sera were tested for latent nuclear and lytic antigens using indirect immunofluorescence and Western blotting. RESULTS: Serology for HHV-8 was positive in 16 of 171 patients (9.35%). Among 13 HIV-positive patients, four (31%) were also positive for HHV-8. The seropositivity among healthy children was 7.6%, and females were significantly (P<0.002) more associated with HHV-8 seropositivity (16%) than males (1.1%). CONCLUSIONS: We detected a higher HHV-8 seroprevalence among the pediatric population in Rio de Janeiro, Brazil, when compared with similar studies performed in other countries. Nonsexual routes of transmission are a possible explanation for this pattern. The unusual finding of a higher seroprevalence among female children could be explained by a specific route of exposition or even an epidemiological bias.

Herpesvírus humano tipo 8(HHV-8): estudo de segmentos alvo do genoma viral em amostras de sangue, saliva e urina de pacientes infectados pelo HIV/aids, com e sem Sarcoma de Kaposi / Human herpesvirus 8 (HHV-8): studies of target segments viral genoma in blood, saliva, and urine from HIV/aids patients infected, with or not Kaposi's sarcoma

Desde a descoberta do herpes vírus humano tipo 8 (HHV-8) como o agente etiológico do sarcoma de Kaposi (SK) nas suas diferentes formas clínico-epidemiológicas, vários estudos vêm sendo conduzidos com o intuito de determinar as vias de transmissão desse vírus em populações endêmicas e de risco epidemiológico. Em regiões endêmicas, a transmissão viral foi relacionada à transmissão horizontal de mães para filhos e entre irmãos e a sexual principalmente, nos casos de SK/aids. Com o objetivo de determinar segmentos do genoma viral em fluídos biológicos e consequentemente seu potencial infectante foi conduzido o presente trabalho. Foram avaliados quanto à presença de segmentos localizados em posições estratégicas do genoma do HHV-8 em sangue, saliva e urina...

Human herpesvirus-8 (HHV-8) antibodies in women from São Paulo, Brazil: association with behavioral factors and Kaposi's sarcoma

BACKGROUND: With the spread of AIDS, many HIV-infected women have been diagnosed with Kaposi's sarcoma (KS), especially in Africa. Since the discovery of a novel herpesvirus as the causative agent of KS (human herpesvirus 8 - HHV-8) several seroepidemiological studies have been conducted to identify groups at risk for KS. The risk for women in Brazil has not been studied. MATERIALS AND METHODS: We searched for HHV-8 antibodies in sera obtained from a bank made up of samples from 3 groups of individuals: Group I: 163 HIV-1-infected women attended at an ambulatory clinic in 1994; Group II: 108 children born to HIV-1-infected mothers from 1990 to 1992, their antibodies reflected maternal infection, and Group III: 630 HIV-1-seronegative, healthy women. In-house immunofluorescence and Western-Blot assays based on the BCBL-1 cell line were used to detect anti-latent and anti-lytic HHV-8 antibodies. RESULTS: Group I had an overall frequency of antibodies of 8.6 percent, with a 1.2 percent frequency of anti-latent antibodies and an 8.0 percent frequency of anti-lytic antibodies. Similar results were detected in Group II, i.e., no cases with anti-latent antibodies and a 7.4 percent frequency of anti-lytic antibodies. In contrast, prevalences of 1.1 percent anti-latent antibodies and 0.3 percent anti-lytic antibodies were observed in Group III. CONCLUSIONS: The epidemiologic pattern of HHV-8 in women from São Paulo varies according to behavioral factors, with emphasis on the sexual and blood routes of virus transmission/acquisition. Although HHV-8 anti-lytic antibodies were found in HIV-1-infected women, no case of KS was detected. Protective factors against KS are probably related to gender and/or to antiretroviral therapies introduced in Brazil since 1994.

Search for virulence-associated antigens of rhodococcus equi in strains isolated from patients with Acquired immunodeficiency Syndrome

Rhodococcus equi (formerly Corynebacterium equi) are known to be highly virulent, intermediate in virulence, or avirulent correlated with specific virulence-associated antigens identified immunochemically by different molecular weights. The association of virulence antigens with infection of AIDS patients by this organism has not been sufficiently evaluated in Brazil or Italy. The objective of the present study was to search for virulence-associated antigens of 15-to 17kD and 20-kD in Rhodococcus equi strains isolated from patients with rhodococcal infection and AIDS. Four Brazilian and 9 Italian strains were studied. All isolates were analyzed by gel electrophoresis followed by immunoblotting using specific monoclonal antibodies to identify virulence-associated antigens. The results obtained on gel electrophoresis analyses showed complexing of R. equi components with proteins of molecular weights ranging from 10-to 150-kD. By immunoblotting, a wide diversity in R. equi virulence-associated antigens was detected: 1 of the 4 Brazilian isolates and 2 Italian isolates had the 15-to 17-kD virulence-associated antigen, 3 Brazilian isolates and 1 Italian isolate had the 20-kD virulence-associated antigen, and the other Italian isolates had no virulence-associated antigens. These results indicate that the pathogenicity of R. equi trains for humans does not depend only on the presence of these well established virulence-associated antigens.

Cytological, cytochemical and immunological findings from two children with chediak-higashi syndrome

This article describes studies of two unrelated patients, aged near 2 years, with leukocyte granulation abnormalities similar to those presented in the Chediak-Higashi Syndrome(CHS). Both patients showed dermatologic manifestations characterized by hypopigmentation of the hair and skin. The cytological and cytochemical study of the peripheral blood leukocytes demonstrated giant abnormal granules with lisosomal content presentd in neutrophils, eosinophils and monocytes. The mononuclear cells displayed one large azurophil granule. The cytochemical functional study of neutrophils showed normal nitroblue-tetrazolium reduction test. The immunological status of the patients were made and in patient 1 was detected a low number of T helper lymphocytes and a maintenance of lymphocytes in continuous cell culture for five weeks without addition of external growth factor. These findings with clinical manifestations permited to establish the CHS, in these cases.

Quantification of lymphocyte subsets in AIDS associated with suspected cytomegalovirus infection

The present study investigates the number of immunoregulatory cells in 55 patients suspected of Acquired Immunodeficiency Syndrone(AIDS) associated with cytomegalovirus(CMV) infection. Patients were divided into three groups: RISK(13 cases), LAS/ARC(21 cases) and AIDS(21 cases) according to epidemiological and clinical findings and using CDC criteria. The CMV infection was suggested based on clinical and serological evaluations. No significant difference in CMV seropositivity was observed among the groups (66,7% to 77,0%), althougha diminished number of cases 3 (14,3%) with CMV-IgM antibody was detected in AIDS group. Phenotypic analysis of leukocytes, lymphocytes and lymphocyte subsets was realized. The results obtained were similar between CMV-seropositive and CMV-seronegative patients in each group studied. On the other hand, the number of T lymphocytes and T helper/inducer lymphocytes were markedly diminished in the LAS/ARC and AIDS groups. The number of T suppressor/cytotoxic lymphocytes was increased in all groups analysed. These alterations in T lymphocyte subsets are responsible for the reversal ratio observed in these patients.