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.

Seroprevalence and molecular epidemiology of human T-Cell leukemia virus type 1 (HTLV-1) and HTLV-2 in blood donors from Dakar, Senegal.

In 2002, human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 seroprevalence was 0.16% (8/4,900) in blood donors from Dakar, Senegal. Most of the positive donors originated from the country's southern region. Seven donors were infected by HTLV-1 (of cosmopolitan subtype), and one was infected by HTLV-2. These data highlight the problem of transfusion safety in this area where HTLV-1-associated lymphoproliferative and neurological diseases are endemic.

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.

[Sero-epidemiological study on the human T-cell leukaemia virus type I/II infection in the east coastal areas of Fujian province].

OBJECTIVE: To study the seroprevalence of human T-cell leukaemia virus type I/II (HTLV-I/II) infection in adult population in the east coastal areas of Fujian and to explore the possible risk factors of HTLV-I/II. METHODS: A total number of 3259 blood samples from drug users, sexually transmitted disease (STD) patients, prostitutes and blood donors for serologic assays during 1999 to 2002, were collected. All samples were screened for HTLV-I/II antibody, using enzyme linked immunosorbent assay (ELISA) kits. All of the positive samples were confirmed by western blot (WB) kits. Statistical analysis was done by Epi software, and chi(2) test by Fisher's exact test. P value < 0.05 was considered statistically significant. RESULTS: The overall seroprevalence rate of HTLV-I/II in healthy populations was 0.06% including, 0.32% in drug users, 0.58% in STD patients and prostitutes respectively. HTLV-II had not been found. The seropositive rates for HTLV-I in STD patients and prostitutes were significantly higher than the findings among healthy populations (P < 0.05). There were no different seroprevalence rates between drug users and healthy populations (P > 0.05). No significant changes in HTLV-I prevalence rates were found in the different age groups as well as in Fuzhou and Linde cities (P > 0.05). CONCLUSION: The result suggested that in the east coastal areas of Fujian province, HTLV-I was the main prevalent virus. The seroprevalence of HTLV-I was very low, with no HTLV-II. Neither age nor gender seemed to be HTLV-I risk factor in the east coastal areas of Fujian province, but the increase of exposure to sex might be one.

Use of a chimeric synthetic peptide from the core p19 protein and the envelope gp46 glycoprotein in the immunodiagnosis of HTLV-II virus infection.

A chimeric synthetic peptide incorporating immunodominant epitope of the p19 gag protein (116-134) and the gp46 env protein (178-200) of HTLV-II virus, separated by two glycine residues, was synthesized by conventional solid-phase peptide synthesis. The antigenic activity of this peptide was evaluated by Ultramicro Enzyme-linked immunosorbent assay (UMELISA) by using panels of anti-HTLV-II positive sera (n = 9), anti-HTLV-I/II positive sera (n = 2), HTLV-positive (untypeable) serum samples (n = 1),and anti-HTLV-I positive sera (n = 14), while specificity was evaluated with samples from healthy blood donors (n = 20). The efficacy of the chimeric peptide in solid-phase immunoassays was compared with the monomeric peptides. Data demonstrated that the chimeric peptide was the most reactive because it detected antibodies to virus efficiently. This may be related to peptide adsorption to the solid surface and epitope accessibility to the antibodies. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of HTLV-II infection.

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.

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.

Frequent detection of antibodies against HTLV antigens in patients with AIDS-related non-Hodgkin lymphoma.

We studied the prevalence of anti-HTLV-I/II antibodies in 22 patients with AIDS-related non-Hodgkin lymphoma (NHL), 453 HIV-1-infected patients without lymphoma (194 of whom were diagnosed as having AIDS), and 6 HIV-1-positive and 75 HIV-1-negative patients with Hodgkin lymphoma. The frequency of serological reactivity against HTLV antigens was significantly higher in the AIDS patients with lymphoma than in those without (8 of 22, 36.4% vs. 20 of 194, 10.3%-p = 0.0027). One of the HIV-1-positive and none of the HIV-1-negative patients with Hodgkin lymphoma showed anti-HTLV-I/II reactivity. Four of the eight seropositive NHL patients showed antibodies directed against HTLV-II recombinant antigens when tested for serological discrimination in a Western blot assay. A PCR study of PBMCs from the only patient with NHL still alive at the time of the study showed HTLV-II-specific sequences in the genomic DNA. These data suggest that HTLV-II or a closely homologous retrovirus infects a high proportion of patients with AIDS-associated NHL.

A novel monoclonal antibody specifically reactive with human T-lymphotropic virus type-II (HTLV-II) envelope protein.

A novel monoclonal antibody (MAb), N5.4.4, specifically reactive with human T-lymphotropic virus type-II (HTLV-II) envelope glycoprotein (gp) has been developed through immunization with a synthetic peptide corresponding to the amino acid sequence 171-196 of the HTLV-II envelope protein (II-env 171-196). This MAb, which belonged to the IgG1 kappa subclass, reacted with the cytosmears of HTLV-II-infected cell lines (Si-IIA, CR-IIA-I and AS-IIA), but not with those of HTLV-I-infected cell lines (MT-1 and MT-2) or other HTLV-uninfected cell lines. On Western blot analysis, this MAb reacted with gp46 of HTLV-II lysates but not with HTLV-1 lysates. Moreover, flow-cytometric analysis revealed that this MAb recognized the native surface of only the HTLV-II-producing cells. Through application to immunohistochemical or serological method, this MAb may be of value in elucidating the pathogenesis of HTLV-II infection in comparison with HTLV-I.

Identification of a type-specific protein that differentiates serologically between human T cell lymphotropic virus types I and II.

The 24-kDa band formed when sera of humans infected with human T cell lymphotropic virus type I (HTLV-1) were reacted with HTLV-I lysates in conventional Western blot (WB) assays was found to be composed of two immunologically unrelated proteins of 24- and 23-kDa. p24, but not p23, carries epitopes shared by the major core proteins of the other known transactivating C-type retroviruses. p23 is unrelated immunologically to the env and tax HTLV-I products but partly cross-reacts with HTLV-I p19. All HTLV-I and simian T cell leukemia virus type I sera tested reacted with p23. Reactivity with p23 was seen with some HTLV-I sera that did not react or reacted weakly with HTLV-I p24. No reactivity with p23 was seen among the 51 human HTLV-II sera tested nor among a large panel of control sera. Because of its type-specificity and strong immunogenicity, p23 provides a reliable serologic marker for the diagnosis of HTLV-I infection and for distinguishing between HTLV-I and -II.

Phenotypic expression of integrin membrane receptors on spontaneously proliferating CD8 cells in human T-lymphotropic virus type II (HTLV-II)-infected individuals.

Spontaneous lymphocyte proliferation in the absence of exogenous stimulators was examined in asymptomatic HTLV-II-seropositive (n = 12) and seronegative individuals (n = 16). Mean spontaneous lymphocytic proliferation significantly increased on day 8 postculture in HTLV-II-infected individuals (5762 +/- 899 cpm) compared with normal controls (2034 +/- 925 cpm, P less than 0.01). The proliferating cells in infected individuals were predominantly T cells; neither B cells nor monocytes demonstrated any proliferation. Phenotypic analysis of cultured cells from individuals with HTLV-II infection demonstrated differential expression of integrin molecules as defined by anti-CD29 and anti-S6F1 (42.8 +/- 4.2 and 39.6 +/- 5.9%, respectively) on CD8 cells, as compared with day 0 peripheral blood mononuclear cells (PBMC) from infected individuals (19.7 +/- 3.5 and 19.9 +/- 1.9%, respectively) or normal controls (12.9 +/- 3.1 and 11.5 +/- 2.5%, respectively; P less than 0.001 for both comparisons). These CD8+ cells did not express CD16 or CD11b. The culture supernatants derived from the spontaneously proliferating cells had significantly increased levels of sCD8 and sCD25 (765 +/- 180 and 1805 +/- 320 U/ml, respectively) compared with those from normal controls (222 +/- 120 and 305 +/- 90 U/ml, respectively; P less than 0.01). Furthermore, culture supernatants derived from spontaneously proliferating PBMC from HTLV-II-infected individuals had no detectable levels of HTLV antigen and did not stimulate proliferation of PBMC from normal donors. These results suggest that the spontaneous proliferation in HTLV-II asymptomatic carriers is due to expansion of CD8 cells expressing integrin receptors which may serve as costimulatory molecules for their activation.

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)

Human T-cell leukemia virus type-I/II (HTLV-I/II) serologic testing: the importance of assaying for the full complement of viral antigens.

Seventy-one Japanese adult T-cell leukemia (ATL) patients and 411 Japanese asymptomatic patients from HTLV-I endemic regions of southern Japan were found to be seropositive by radioimmunoprecipitation assay (RIPA). Of these 482 positive controls, 62% of ATL patients and 67% of the asymptomatic seropositive patients were found to harbor antibodies to p40x. Additionally, 333 preselected Japanese blood donors who were identified as seropositive by particle agglutination (PA) assay were further tested for antibodies to HTLV-I/II gene encoded envelope (env) or group specific antigens (gag) by means of enzyme-linked immunosorbent assay (ELISA) and RIPA. Concordance between ELISA and RIPA was noted in 318 samples (92.5%). Discordance between ELISA and RIPA was observed in 15 sera (7.5%)--2 were seropositive by ELISA and seronegative by RIPA and 13 were seronegative by ELISA and seropositive by RIPA. Seven of these 13 samples (53.8%) contained antibodies to p40x by RIPA and may represent ELISA false negatives on the basis of both clinical and laboratory data. Current HTLV-I/II ELISA kits may yield false negative results. Additional research into the development of rapid detection cost-efficient assays that test for the full compliment of viral antigens is needed.

Identification of immunodominant epitopes in envelope glycoprotein of human T lymphotropic virus type II.

A series of synthetic peptides derived from the envelope glycoprotein of human T lymphotropic virus type II (HTLV-II) was used in an enzyme immunoassay to determine the immunodominant epitopes of envelope glycoprotein. Of the 11 synthetic peptides spanning the external glycoprotein of HTLV-II (gp52) and the 3 from the transmembrane protein (gp21), 3 peptides from gp52 (termed Env-20(85-102), Env-202(173-209), and Env-203(219-256] reacted with most of the polymerase chain reaction-confirmed HTLV-II specimens (83, 95, and 76%, respectively); all other peptides reacted minimally with these specimens. Env-202(173-209) reacted with a greater percentage (91 to 100%) of specimens from different risk groups, including intravenous drug users (n = 30), North American Indians (n = 13), Guaymi Indians from Panama (n = 22), and routine U.S. blood donors (n = 34), when compared with Env-20(85-102) (73 to 100%) or Env-203(219-256) (68 to 83%). Furthermore, Env-20(85-102) and Env-202(173-209) had some reactivity (8-25%) with sera from HTLV-I-infected individuals, whereas Env-203(219-256) reacted with 58% of HTLV-I specimens. We conclude that peptides Env-20(85-102) and Env-202(173-209) represent the type-specific immunodominant epitopes of HTLV-II external glycoprotein.

Serological discrimination between HTLV-I and HTLV-II antibodies by ELISA using synthetic peptides as antigens.

Using the peptides from amino acids 100-130 of the HTLV-I gag protein, 175-199 of the HTLV-I env protein and the corresponding peptides of HTLV-II (amino acids 106 to 135 of the gag protein and 171 to 196 of the env protein), we tested for reactivity against antibodies by enzyme immunoassay in sera from HTLV-I and HTLV-II carriers. The peptides derived from the env proteins have high specificity for antibody binding. The peptide based on amino acids 175-199 of HTLV-I reacted with antibodies in sera from all HTLV-I carriers, and the peptide composed of amino acids 171-196 of HTLV-II reacted with antibodies in sera from all HTLV-II carriers. For the peptides derived from the gag proteins, we observed some cross-reactivity in sera from persons with anti-HTLV-I and anti-HTLV-II, due to antibody binding to the peptide corresponding to 12 amino acids from the C-terminal end of the gag protein. Separate enzyme immunoassays that used the four synthetic peptides as antigens clearly distinguished between serum with antibodies to HTLV-I or HTLV-II in various individuals and excluded false positive results using the particle agglutination assay that used a whole-virus lysate of HTLV-I as antigen.