Comparison of four commercial screening assays for the diagnosis of human T-cell lymphotropic virus types 1 and 2.

Serological assays for human T-cell lymphotropic virus types 1 and 2 (HTLV-1/2) are widely used in routine screening of blood donors. The aim of this study was to compare the performance of four commercial screening assays for HTLV-1/2 infection frequently used in South America. A total of 142 HTLV-1 and HTLV-2 seropositive and 336 seronegative samples were analyzed by using four commercial tests (BioKit, Vironostika, Murex and Fujirebio). These tests are commonly used for HTLV-1/2 detection in blood banks in Argentina. A nested-PCR was used as the reference standard. The most sensitive tests for HTLV-1/2 were Fujirebio and Biokit (98.6%) followed by Murex (97.2%) and Vironostika (96.5%). The most specific test was Murex (99.7%), followed by Biokit (97.0%), Fujirebio (95.8%), and Vironostika (92.9%). The kappa index of agreement was higher for Murex (kappa=0.97), followed by BioKit (kappa=0.94), Fujirebio (kappa=0.92), and Vironostika (kappa=0.86). The highest index of agreement was shown by Murex test while Vironostika had the lowest performance. Of the four tests evaluated, only the Vironostika assay is approved by the Food and Drug Administration. These results should be considered for choosing the most accurate serological screening assays in order to obtain an optimal efficiency of the current algorithm for HTLV-1/2 diagnosis.

Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

Antigenic mixture of synthetic peptides for the immunodiagnosis of HTLV I/II infection.

Monomeric and chimeric synthetic peptides were used as coating antigens in four different mixtures in a solid phase immunoassay to select an optimal combination for the detection of antibodies to human T-cell lymphotropic virus (HTLV) in serum samples. The peptides, P-13 (gp21 I), Q5 (gp21 II)-GG-(gp46 II), and Q (gp46 I)-GG-(p19 I), represent immunodominant sequences from transmembrane protein (gp21), envelope protein (gp46), and core protein (p19) of HTLV I/II viruses; they were the most antigenic and specific peptides in previous studies. The sequences of the chimeric peptides were separated by two glycine residues. An indirect UltramicroEnzyme-linked immunosorbent assay (UMELISA) was used to evaluate the antigenicity of these peptide mixtures by using samples from anti-HTLV I/II PRP205(M), (n = 20), HTLV I-infected individuals from Cuba (n = 7), and HTLV I-positive sera from Colombia and Chile (n = 9). The specificity was evaluated with healthy blood donor sera (n = 300), anti-HIV 1-positive samples (n = 10), and other seropositive samples to different infectious agents. The highest sensitivity and specificity was obtained with mixture 1, which could be very useful in the immunodiagnostic of HTLV infection.

Pathological and virological assessment of acute HTLV-I-associated myelopathy complicated with encephalopathy and systemic inflammation.

HTLV-I-associated myelopathy, also known as tropical spastic paraparesis (HAM/TSP), is a chronic inflammatory disease of the spinal cord. Acute cases are uncommon. We report the case of a 41-year-old woman with acute HAM/TSP complicated with encephalitis, an intense inflammatory reaction of the nervous system and lymphocytic infiltration of skeletal muscles, liver, salivary, adrenal and pituitary glands. The immunohistochemical studies of the lymphocytes surrounding blood vessels showed both B- and T-lymphocytes, in similar proportion, with both CD4- and CD8-positive cells. In addition, many perivascular and scattered macrophages were observed. Adult T-cell leukemia/lymphoma (ATL) was ruled out. The marrow aspirate was normal. Serial cerebrospinal fluid (CSF) analysis showed presence of HTLV-I antibodies, but without intrathecal synthesis of specific antibodies. Determination of HTLV-I viral loads demonstrated increased levels in the CSF relative to the peripheral blood and may be associated with widespread inflammation. The pathological and immunological findings may help understand the role of immune-reactive cells in the pathogenesis of HTLV-I-associated myelopathy.

Antigenicity of chimeric synthetic peptides based on HTLV-1 antigens and the impact of epitope orientation.

The present study evaluated four chimeric synthetic peptides incorporating immunodominant sequences from HTLV-1 virus. Monomeric peptides M1, M2, and M3 represent sequences from core (p19) and envelope (gp46) of the virus. The peptide M1 is a p19 (105-124) sequence, the peptide M2 is a gp46 (190-207) sequence, and the peptide M3 is a gp 46 sequence with substitution of proline at position 192 by serine. Those peptides were arranged in such a way that permits one to obtain different combinations of chimeric peptides (M1-M2, M2-M1, M1-M3, and M3-M1). Two glycine residues were used as arm spacers for separating the two sequences. The antigenicity of these peptides was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of human T cell leukemia virus type I (HTLV-I)-infected individuals (n = 24), while specificity was evaluated with anti-HTLV-II-positive samples (n = 11) and healthy blood donors (n = 25). The results were compared to plates coated with monomeric peptides M1, M2, and M3. The chimeric peptide orientation (M1-M2) and the proline at position 192 of the gp46 peptide showed higher sensitivity.

Human T-cell lymphotropic virus type 1 infection and ocular manifestations in São Paulo, Brazil.

OBJECTIVES: Although human T-cell lymphotropic virus type 1 (HTLV-1)-associated uveitis has been well recognized in Japan, related studies in Brazil are scarce. We performed a serologic survey for HTLV-1 infection among patients with uveitis and investigated the ocular findings in HTLV-1-asymptomatic carriers. METHODS: One hundred ninety serum samples from patients with uveitis of determined (n = 137) and undetermined origins (n = 53) being examined at the Uveitis Service, University of São Paulo, São Paulo, Brazil, underwent testing using HTLV enzyme-linked immunosorbent assay and discriminatory Western blots. One hundred five asymptomatic blood donors and/or their relatives who were seropositive for HTLV-1 (carrier group) and 105 age- and sex-paired blood donors who were seronegative for HTLV-1 (control group) underwent ocular evaluation. For the statistical analysis, chi2 test was used. RESULTS: Only 1 patient with uveitis was seropositive for HTLV- 1, and she belonged to the group with uveitis of undetermined origin. Results of tear films were evaluated in 52 carriers. The prevalence of a decreased tear break-up time was significantly higher in the carrier compared with the control group (P = .02). Two carriers had keratoconjunctivitis sicca. Three of the 105 carriers exhibited mild uveitis (cells in the vitreous, retinal and choroidal infiltrates, retinal vasculitis, and bilateral pars planitis). Retinal pigmentary changes were found in both groups (no statistical difference). CONCLUSIONS: Early tear abnormalities may be present in asymptomatic carriers, and mild uveitis may be found among them. The relatively low seroprevalence of HTLV-1 in the Brazilian population made it difficult to establish the real importance of HTLV-1-associated uveitis among our patients with uveitis.

Laboratory characterization of human T cell lymphotropic virus types 1 (HTLV-1) and 2 (HTLV-2) infections in blood donors from Sao Paulo, Brazil.

Serologic screening for human T cell lymphotropic virus types 1/2 (HTLV-1/2) infection in blood donors has been recently introduced in Brazil. Analysis of 351,639 blood donations in Sao Paulo from January 1992 to October 1993 identified 1,063 positive (0.30%) and 2,238 indeterminate (0.63%) samples based on serologic confirmation using a 21e Western blot. A detailed analysis (serologic, molecular, and virologic), based on a laboratory diagnostic algorithm for characterization of HTLV-1 and HTLV-2 infections was undertaken in 50 seropositive or seroindeterminate blood donors. Modified serologic assays (2.3 Western blot that incorporate type-specific recombinant peptides) performed in 29 HTLV-1/2 positive and 21 HTLV-1/2 indeterminate donors with the 21e Western blot identified 25 as infected with HTLV-1, four with HTLV-2, five with untypable HTLV-1/2, 15 as HTLV-1/2 indeterminate, and one as seronegative. Polymerase chain reaction (PCR) analysis using DNA amplification of proviral pol and tax sequences from peripheral blood mononuclear cells confirmed HTLV-1 and HTLV-2 infections in all 2.3 Western blot seropositive donors; of the five serologically untypable donors, three were confirmed to be HTLV-1 positive, one HTLV-2 positive, and one negative by PCR. All of the seroindeterminate donors were also negative by PCR. Furthermore, HTLV-1 could be isolated in cocultures from 10 of 18 infected donors. Cell lines developed from two HTLV-1-infected donors were of T cell phenotype (CD2+, CD3+), exhibiting surface markers of activated CD4 cells (CD4+ CD25+ HLA-DR+). Thus, we provide evidence for the high seroprevalence of HTLV infection in blood donor population in Sao Paulo, Brazil compared with North American donors and propose a comprehensive serologic and genotypic diagnostic algorithm for HTLV-infected donors that has strong implications for counseling of these individuals.

PIP: Blood donors in Brazil have recently begun to be screened for infection with HTLV types 1 and 2. Of 351,639 blood donations screened in Sao Paulo from January 1992 to October 1993, 1063 positive and 2238 indeterminate samples were identified based upon serologic confirmation using the 21e Western blot. Detailed serologic, molecular, and virologic analysis, based upon a laboratory diagnostic algorithm for the characterization of HTLV-1 and HTLV-2 infections, was conducted upon 50 seropositive or seroindeterminate blood donors. 2.3 Western blot serologic assays, which incorporate type-specific recombinant peptides, performed in 29 HTLV 1/2 positive and 21 HTLV 1/2 indeterminate donors with the 21e Western blot identified 25 as infected with HTLV-1, 4 with HTLV-2, 5 with untypeable HTLV 1/2, 15 as HTLV 1/2 indeterminate, and 1 as seronegative. Polymerase chain reaction (PCR) analysis using DNA amplification of proviral pol and tax sequences from peripheral blood mononuclear cells confirmed HTLV-1 and HTLV-2 infections in all 2.3 Western blot seropositive donors. Of the 5 serologically untypeable donors, 3 were found to be HTLV-1-positive, 1 HTLV-2-positive, and 1 negative by PCR. All seroindeterminate donors were also negative by PCR. HTLV-1 could be isolated in cocultures from 10 of 18 infected donors.

Specificity of intrathecal IgG synthesis for HTLV-1 core and envelope proteins in HAM/TSP.

INTRODUCTION: In patients with human T-cell lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP), we investigated the significance of HTLV-1 specific antibodies in the cerebrospinal fluid (CSF). MATERIAL AND METHODS: The quantities of HTLV-1 specific immunoglobulin G (IgG) in paired CSF and serum were evaluated by a sensitive enzyme immunoassay (EIA). The specificity of antiviral IgG was determined by radioimmunoprecipitation of HTLV-1 antigens. RESULTS: In 17 of 20 HAM/TSP patients, quantitative evaluation by EIA supplied evidence for antiviral IgG synthesis within the CNS. Radioimmunoprecipitation demonstrated IgG antibodies against HTLV-1 envelope and core proteins in all HAM/TSP CSF and sera tested. Regarding the 3 sample pairs indeterminate in EIA for intrathecal synthesis, 2 showed stronger precipitation of HTLV-1 antigens by CSF IgG than by equal amounts of serum IgG. CONCLUSION: Intrathecal antibody synthesis specific for both HTLV-1 core and envelope antigens is common in HAM/TSP, thus providing conclusive evidence for an immune response to HTLV-1 within the CNS.

Mother-to-child transmission of human T-cell lymphotropic virus type I (HTLV-I) in Jamaica: association with antibodies to envelope glycoprotein (gp46) epitopes.

To study mother-to-child transmission of HTLV-I in Jamaica, we screened antenatal patients in Kingston, Jamaica, from 1983 to 1985. Of 2,329 women, 81 (3.5%) were HTLV-I seropositive. Two to three years later, 36 seropositive mothers were recontacted, and blood was drawn from them and their children. All sera were tested for HTLV-I antibodies, and mother's sera were additionally tested for HTLV-I whole-virus antibody titer, syncytium-inhibition neutralizing antibody titer, and titers to six synthetic peptides from the HTLV-I envelope glycoprotein gp46. Seventeen of 74 (23%) [95% confidence interval (CI) 15-34%] children were seropositive. HTLV-I transmission was associated with breast-feeding duration > 6 months [relative risk (RR) 3.2; CI 0.4-22.1], maternal age > 30 years (RR 2.8; CI 1.0-7.8), and higher maternal whole-virus antibody titer (RR 3.3; CI 1.3-8.5). After controlling for higher whole-virus antibody titer, transmission remained associated with higher titer of neutralizing antibody and higher titer of antibody to the peptide sp4a1, corresponding to amino acids 196-209 of the gp46 envelope glycoprotein. We conclude that mother-to-child transmission of HTLV-I in Jamaica is associated with longer duration of breast-feeding, older age, and higher HTLV-I antibody titer, in particular to a certain immunogenic portion of the gp46 envelope glycoprotein.

Mother-to-child transmission of human T-cell lymphotropic virus type I (HTLV-1) in Jamaica: association with antibodies to envelope glycoprotein (gp46) epitopes

To study mother-to-child transmission of HTLV-I in Jamaica, we screen antenatal patients in Kingston, Jamaica, from 1983 to 1985. Of 2,329 women, 81 (3.5 percent) were HTLV-I seropositive. Two to three years later, 36 seropositive mothers were recontacted, and blood was drawn from them and their children. All sera were tested for HTLV-I antibodies, and mother's sera were additionally tested for HTLV-I whole-virus antibody titer, syncytium-inhibition neutralizing antibody liter, and titers to six synthetic peptides from the HTLV-I envelope glycoprotein gp46. Seventeen of 74 (23 percent) [95 percent confidence interval (CI) 15-34 percent] children were seropositive. HTLV-I transmission was associated with breast-feeding duration > 6 months [relative risk (RR) 3.2; CI 0.4-22.1], maternal age > 30 years (RR 2.8; CI 1.0 - 7.8), and higher maternal whole-virus antibody titer (RR 3.3; CI 1.3 - 8.5). After controlling for higher whole-virus antibody titer, transmission remained associated with higher titer of neutralizing antibody and higher titer of antibody to the peptide sp4al, corresponding to amino acids 196 - 209 of the gp46 envelope glycoprotein. We conclude that mother-to-child transmission of HTLV-I in Jamaica is associated with longer duration of breast-feeding, older age, and higher HTLV-I antibody titer, in particular to a certain immunogenic portion of the gp46 envelope glycoprotein.(AU)

Segregation of human T cell lymphotropic virus type I and II infections by antibody reactivity to unique viral epitopes.

A recombinant protein of the human T cell lymphotropic virus type I (HTLV-I) gp46 outer membrane envelope, MTA-4 (residues 129-203), reacted by Western blot with sera from HTLV-I-infected individuals from the United States and Jamaica but not with 24 (10%) of 242 Japanese sera. A related gp46 recombinant protein, MTA-1 (residues 162-209), reacted with all 58 sera from HTLV-I-infected US and Jamaican individuals and 238 of 242 sera from infected Japanese (combined sensitivity of 99%). Neither recombinant showed reactivity to sera from HTLV-II-infected individuals or uninfected controls. The reactivity of recombinant proteins containing the region of HTLV-II gp46 analogous to MTA-1 was also evaluated by Western blot: GH2-K15 (residues 157-205) and GH2-K55 (residues 162-205) reacted with 88 (98%) and 89 (99%), respectively, of 90 sera from HTLV-II-infected individuals but not with sera from HTLV-I-infected individuals or uninfected controls. These recombinant proteins should permit the development of assays to unambiguously confirm and differentiate HTLV-I and HTLV-II infections.

Segregation of human T cell lymphotropic virus type I and II infections by antibody reactivity to unique viral epitopes

A recombinant protein of the human T cell lymphotropic virus type I (HTLV-I) gp46 outer membrane envelope, MTA-4 (residues 129-203), reacted by Western blot with sera from HTLV-I-infected individuals from the United States and Jamaica but not with 24 (10 percent) of 242 Japanese sera. A related gp46 recombinant protein, MTA-1 (residues 162-209), reacted with all 58 sera from HTLV-I-infected US and Jamaican individuals and 238 of 242 sera from infected Japanese (combined sensitivity of 99 percent). Neither recombinant showed reactivity to sera from HTLV-II-infected individuals or uninfected controls. The reactivity of recombinant proteins containing the region of HTLV-II gp46 analogous to MTA-1 was also evaluated by Western blot: GH2-K15 (residues 157-205) and GH2-K55 (residues 162-205) reacted with 88 (98 percent) and 89 (99 percent), respectively, of 90 sera from HTLV-II-infected individuals but not with sera from HTLV-I-infected individuals or uninfected controls. These recombinant proteins should permit the development of assays to unambiguously confirm and differentiate HTLV-I and HTLV-II infections. (AU)