Differential antibody responsiveness to p19 gag results in serological discrimination between human T lymphotropic virus type I and type II.

A new algorithm based upon the differential antibody responses to two gag gene products (p19 and p24) of human T lymphotropic virus (HTLV) has been suggested for serologic discrimination of HTLV type I (HTLV-I) and type II (HTLV-II) [Lillihoj et al., 1990]. To evaluate the practical usefulness of this algorithm, serum specimens from HTLV-seropositive individuals whose infection was confirmed by PCR analysis to be HTLV-I (n = 60) or HTLV-II (n = 61) were analyzed by western blot. The intensities of the antibody response to p24gag and p19gag were scored by one individual without prior knowledge of PCR results. According to the algorithm, specimens with p19 greater than or equal to p24 were classified as HTLV-I, whereas specimens with p19 less than p24 were classified as HTLV-II. Of 60 PCR confirmed HTLV-I specimens, 56 had p19 greater than or equal to p24 (93%) while 4 had p19 less than p24. Of 61 PCR confirmed HTLV-II specimens, 56 had p19 less than p24 (92%) and 5 had p19 greater than or equal to p24. The overall accuracy of serologic differentiation when using this algorithm was 92%, as 4 of 60 HTLV-I (7%) and 5 of 61 HTLV-II (8%) could have been wrongly classified. Although the differential antibody response to p19gag and p24gag provides a simple means of serologically distinguishing between HTLV-I and HTLV-II infection in population-based epidemiological studies, in a clinical context more accurate means of confirmation are required. The dominant p19gag responses were mapped to the C-terminus of p19 (p19(102-117)).(ABSTRACT TRUNCATED AT 250 WORDS)     

Human T-cell lymphotropic virus types I and II infections in patients with leukaemia/lymphoma and in subjects with sexually transmitted diseases in Nigeria

Summary Serological assays that distinguish antibodies to human T-cell lymphotropic virus types I (HTLV-I) and type II (HTLV-II), and polymerase chain reaction (PCR) tests were used to investigate association of these two human retroviruses with several well-defined clinical conditions in Nigeria. We compared the frequency of HTLV-I and HTLV-II infections among patients with lymphoproliferative disorders (n=65), individuals with various sexually transmitted diseases (n=40), patients with genital candidiasis (n=25) and apparently healthy individuals (n=60). Serological analysis of blood samples from all four groups showed that 10 of the 190 (5.3%) individuals tested were confirmed positive for the presence of antibodies to HTLV-I (6) or HTLV-II (4). Using the PCR technique, specific HTLV-I or HTLV-II sequences were amplified from the genomic DNA of 4 of 6 HTLV-I seropositive and 3 of the 4 HTLV-II seropositive individuals respectively. However, sequences of both viruses were amplified from the genomic DNAs of the remaining 3 seropositive individuals. Since one of the 5 sets of primer pairs [(SK110 (II)/SK III (II)], which is used for specific identification of HTLV-II did not amplify the target sequence from the genomic DNAs of any of the 4 HTLV-II-confirmed seropositive individuals in this study, it suggested sequence diversity of these viruses in Nigeria. The virus-infected individuals identified in this study were one (1.5%) of the 65 patients with leukaemia/lymphoma (HTLV-I), 6 of 40 (15.0%) individuals (HTLV-I=1, HTLV-II=3, HTLV-I/II=2) with sexually transmitted diseases (STD), one of 25(4.0%) subjects with genital candidiasis for HTLV-I, and 2 of 60 (33.3%) healthy individuals (one for HTLV-I and one for HTLV-I/II). There was a significant difference (P<0.025) between the prevalence of HTLV-I/II infections among patients with lymphoma/leukaemia and those who attended STD clinic in Ibadan, Nigeria. This study also suggests that while HTLV-I and HTLV-II may be important sexually transmitted viruses, they may not be specific aetiological agents of the common lymphoproliferative disorders in Nigeria.     

Pooling of samples for seroepidemiological surveillance of human T-cell lymphotropic virus types I and II

We evaluated a straight forward pooling strategy for antibody screening of HTLV-I/II, using panels of sera from various parts of the world including a total of 43 HTLV-I and 54 HTLV-II positive specimens. Four antibody screening assays were included in the evaluation: the HTLV-I/II GE 80/81 (Murex Diagnostics), the HTLV-I/HTLV-II Ab Capture ELISA (Ortho Diagnostics), the HTLV-I/II ELISA 3.0 (Genelabs Diagnostics) and the Serodia HTLV-I (Fujirebio). The Murex and Ortho assays represent a new generation of HTLV screening tests with a sandwich format incorporating both HTLV-I and HTLV-II synthetic and/or recombinant peptide antigens. The Genelabs assay is an indirect ELISA with recombinant HTLV-I and -II antigens and Serodia is a particle agglutination assay with HTLV-I whole viral lysate. Each HTLV-positive sample was included in pools of 1/1 up to 1/16, in two-fold steps made in normal HTLV-negative blood donor serum from one up to nine donors. For HTLV-I, with the exception of one false negative sample in dilution 1/16 with Genelabs ELISA, all assays were positive at all dilutions. The Murex assay had absorbance values at maximum levels for all samples at all dilutions. The other assays had gradually decreasing absorbance values although clearly above cut-off. For HTLV-II, the Murex assay correctly detected all samples to dilution 1/16 despite gradually decreasing signals. The Serodia assay had 100% sensitivity to dilution 1/4 while at 1/8 and 1/16 it decreased 82 and 80%, respectively. The Genelabs ELISA had gradually decreasing sensitivity for HTLV-II from 98 (1/1) to 33% (1/16) while the Ortho assay detected all specimens at all dilutions in a limited set of samples tested. Taken together, this evaluation has shown that pooling of samples may be an appropriate strategy for serosurveillance of HTLV. It is, however, crucial to limit the number of samples and to choose assays that allow the dilution caused by the pooling. Using the best performing assays in this evaluation for pools of e.g. five samples would leave a reasonable safety margin.     

Discrimination between human T-cell lymphotropic virus type I and II (HTLV-I and HTLV-II) infections by using synthetic peptides representing an immunodominant region of the core protein (p19) of HTLV-I and HTLV-II.

We describe enzyme immunoassays that use synthetic oligopeptides to discriminate serologically between human T-cell lymphotropic virus type I and II (HTLV-I and HTLV-II) infections. The peptides represented 20-amino acid segments between residues 111 and 130 (MA1) and residues 116 and 135 (MA2) of the p19 gag proteins of HTLV-I and HTLV-II, respectively. The assays were sensitive since 69 of 74 HTLV-positive sera were reactive to at least one of the two matrix (MA) peptides (sensitivity, 93.2%). By using the ratio of the optical density of MA1 to the optical density of MA2, which represents for every serum sample the ratio between the absorbance value obtained in the MA1 assay and the absorbance value obtained in the MA2 assay, 59 of the 69 reactive serum samples were clearly and easily typed as positive for either antibody to HTLV-I or antibody to HTLV-II. Eight of the 10 remaining reactive serum samples were analyzed further by an inhibition procedure, and their type specificities were then clearly identifiable. Therefore, the results indicate that all MA-reactive sera were serologically distinguished by our peptide assays.     

Comparison of immunofluorescence, enzyme immunoassay, and Western blot (immunoblot) methods for detection of antibody to human T-cell leukemia virus type I.

A total of 3,349 serum samples were screened by the immunofluorescence (IF) method for antibody to human T-cell leukemia virus type I (HTLV-I). Only 9 of 2,409 specimens from selected individuals, blood bank donors, patients with encephalitis-meningitis, and human immunodeficiency virus antibody-positive homosexual or bisexual men were reactive by IF. In addition, 940 serum samples from intravenous drug abusers were tested by IF and also by an HTLV-I enzyme immunoassay (EIA) method. Of these, 222 (24%) were positive for both HTLV-I and HTLV-II antigens by IF, and 191 of these 222 were also reactive in the HTLV-I EIA. Of the 31 IF-positive, EIA-negative serum samples, 20 exhibited optical density readings greater than or equal to 70% of the positive cutoff in the EIA, and 29 samples reacted with 1 or more bands in the Western blot (immunoblot) test. An additional 10 specimens that were EIA negative reacted only with HTLV-I by IF. Differences in staining morphology and in reactions on HTLV-I and HTLV-II antigens before and after absorption of the serum specimens with HTLV-I and HTLV-II-infected cell pellets revealed six distinct serological patterns by IF. These results indicate that infections by HTLV-I or by another closely related retrovirus(es) occur in California. Further studies utilizing statistically valid sampling methods are needed to estimate true prevalence rates among various groups. IF and Western blot tests should supplement the EIA method to maximize sensitivity and specificity of test procedures.     

Detection of antibodies to trans-activator protein (p40taxI) of human T-cell lymphotropic virus type I by a synthetic peptide-based assay.

Antibodies to human T-cell lymphotropic virus type I (HTLV-I) trans-activator protein (p40taxI) were determined in serum specimens from individuals infected with HTLV-I (n = 138) and HTLV-II (n = 19). Western blot (immunoblot) analysis using recombinant tax demonstrated the presence of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-I-associated myelopathy, 43% of those (20 of 46) with adult T-cell leukemia, and 61% of asymptomatic HTLV-I blood donors (40 of 66); only one of the HTLV-II specimens reacted with the recombinant tax protein. Synthetic peptides (Tax8(106-125), Tax22(316-335), Tax-23(331-350), and Tax-24(336-353) representing the immunodominant epitopes of¿ p40taxI detected anti-tax antibodies in 66 (48%), 50 (36%), 66 (48%), and 64 (46%) of 138 HTLV-I-positive specimens, respectively. An enzyme immunoassay using an equimolar ratio of these four peptides allowed sensitive detection of anti-tax antibodies in 96% of patients (25 of 26) with HTLV-1-associated myelopathy, 52% of adult T-cell leukemia patients (24 of 46), and 62% of asymptomatic HTLV-1-infected donors (41 of 66). The synthetic peptide-based cocktail assay was HTLV-I specific, since none of the HTLV-II-infected specimens reacted with these peptides. Interestingly, the corresponding regions from the HTLV-II tax protein, Tax8II(106-125), and Tax-22II(312-331) did not react with either HTLV-II or HTLV-I specimens. Thus, a synthetic peptide-based assay composed of immunodominant epitopes located towards the amino terminus and the C terminus of p40taxI provides a reliable and sensitive assay for the detection of anti-tax antibodies in seroepidemiologic studies.     

Serological speciation of human T-cell leukaemia virus infections using synthetic peptide antigens

In order to assess the specificity and sensitivity of two peptide-based assays (Synth^TM HTLV-I and HTLV-II enzyme-linked immunoassay [EIA] [UBI] and Select-HTLV^TM EIA [IAF]) in discriminating between antibody to HTLV-I and HTLV-II infection, a panel of 186 well-characterised serum/plasma samples was tested by the two assays. The panel comprised 160 samples that by Western blot were confirmed to contain antibodies to HTLV-I/II and 26 samples that showed reactivity with gag but not env gene products. Both assays were found to be specific in that they did not misclassify any of the 80 specimens from cases of tropical spastic paraparesis or adult T-cell leukaemia/lymphoma, diseases believed to be HTLV-I associated, as anti-HTLV-II positive. Of the 160 specimens confirmed as anti-HTLV-I/II positive by Western blot, 6.2% were negative or untypable in the Synth EIA compared with 13.7% in the Select EIA. Of the 26 Western blot indeterminate samples, 16 were negative by both assays. Five were typed as anti-HTLV-I by both assays and 5 as anti HTLV-II by Select EIA only. The peptide based EIAs offer an economical and, in most cases, reliable means of discriminating between anti-HTLV-I and anti-HTLV-II. However, they should only be applied to sera that have been confirmed by Western blot or other methods as anti-HTLV-I/II positive. Even then they may fail to speciate sera from non-Japanese, non-Afrocaribbean populations. © 1993 Wiley-Liss, Inc.     

Characterization of antibody reactivity to human T-cell lymphotropic virus types I and II using immunoblot and radioimmunoprecipitation assays.

We have characterized the immunoreactivity to human T-cell lymphotropic virus type I (HTLV-I) among 26,983 persons of various seroprevalence groups by using enzyme immunoassay, immunoblot (IB), and radioimmunoprecipitation assays (RIPA) in accordance with Public Health Service recommended guidelines for the interpretation of serologic test results for HTLV-I infection. IB-indeterminate serum specimens (n = 178) were reactive to HTLV-I gag proteins, and no serum contained only env reactivity. Overall, RIPA resolved 40% of IB-indeterminate serum samples; however, the probability that RIPA would confirm IB-indeterminate samples depended on the seroprevalence of the population tested. HTLV-I gag p19-only reactivity on IB was not a reliable marker of HTLV-I infection, while gag p24 reactivity on IB was clearly associated with positive seroreactive specimens. IB and RIPA tests did not clearly distinguish between HTLV-I and HTLV-II seroreactivities. These data emphasize that patterns of immunoreactivity to HTLV-I antigens are dependent upon the seroprevalence of the risk groups tested. In addition, RIPA detected antibodies to env proteins present in low titer in a substantial number of IB gag-only reactive sera and resolved the HTLV-I antibody status of these sera.     

Immunologic cross-reactivity between structural proteins of human T-cell lymphotropic virus type I and the blood stage of Plasmodium falciparum.

To determine the serologic cross-reactivity between human T-cell lymphotropic virus type I (HTLV-I) and parasite antigens, we measured antibody responses against HTLV-I, Plasmodium falciparum, Plasmodium vivax, and Brugia malayi in serum specimens obtained from regions where malaria (n = 482) and filariasis (n = 101) are endemic. Analysis of immune reactivity to HTLV-I antigens showed that specimens from regions where malaria is endemic had significantly higher rates of enzyme immunoassay (EIA) reactivity (76 of 482 [15.8%] than those from regions where filariasis is endemic (0 of 101 [0%]). Western blot (immunoblot) analysis of the HTLV-I EIA-reactive specimens demonstrated predominant Gag reactivity (HTLV-Iind). Only two specimens each from Indonesia and Brazil and four specimens from Papua New Guinea had Env reactivity by radioimmunoprecipitation analysis. Furthermore, a positive correlation between HTLV-EIA and titers of antibody to the blood stage of P. falciparum (rs = 0.24, P < 0.005) was discerned; no correlation was observed between antibodies to the blood stage or the circumsporozoite protein of P. vivax and the circumsporozoite protein of P. falciparum. In addition, P. falciparum-infected erythrocyte lysate specifically abrogated binding of Gag-specific antibodies in HTLV-Iind specimens from regions where malaria is endemic without affecting binding in HTLV-I-seropositive specimens, suggesting that the immunologic cross-reactivity between HTLV Gag proteins and malaria parasites is restricted to the blood-stage antigens of plasmodia in specimens from regions where malaria is endemic. However, HTLV-seroindeterminate specimens from the United States did not demonstrate serologic cross-reactivity, suggesting that antigenic mimicry of HTLV proteins extends to other nonplasmodial antigens as well.     

Development and evaluation of a human T-cell leukemia virus type I serologic confirmatory assay incorporating a recombinant envelope polypeptide.

A recombinant protein derived from the gp21 region of the human T-cell leukemia virus type I (HTLV-I) env gene was synthesized in Escherichia coli and purified by reversed-phase high-performance liquid chromatography. The purified protein was free of contaminating bacterial proteins and retained reactivity with human HTLV-I- and HTLV-II-positive sera and a gp21 monoclonal antibody. An immunoblot procedure using the recombinant polypeptide in conjunction with native viral proteins was more sensitive than the conventional immunoblot and radioimmunoprecipitation confirmatory assays for detection of antibodies to HTLV-I and HTLV-II env-encoded gene products. The recombinant protein was equally reactive with sera from polymerase chain reaction-confirmed HTLV-I or HTLV-II infections. Furthermore, on the basis of the differential reactivities of gp21-positive sera with the HTLV-I p19 and p24 gag-encoded proteins, an algorithm was proposed to distinguish exposure to HTLV-I from exposure to HTLV-II. These results establish the utility of a modified immunoblot assay incorporating a recombinant envelope polypeptide as an alternative to existing HTLV-I-confirmatory assays.     

Serologic confirmation of simian T-lymphotropic virus type I infection by using immunoassays developed for human T-lymphotropic virus antibody detection.

Serum specimens from diverse species of Old World monkeys, categorized as seropositive (n = 97) or seronegative (n = 23) for human T-lymphotropic virus (HTLV) infection, were tested by using recombinant env-spiked Western immunoblot assays and synthetic peptide assays for simultaneous detection and discrimination of simian T-lymphotropic virus (STLV) infection. Of the 97 seropositive specimens, 93 reacted with the recombinant transmembrane (r21env) protein and 90 reacted with a recombinant, MTA-1, derived from the central region of the external glycoprotein of HTLV-I (rgp46env), thus yielding test sensitivities of 96 and 93%, respectively. While 1 of the 23 negative monkey specimens reacted with r21env, none reacted with rgp46env, for overall specificities of 96 and 100%, respectively. Analysis of synthetic peptide-based immunoassays demonstrated that while 85 of 97 (88%) seropositive specimens reacted with HTLV-I-specific epitope (p19gag), none of the specimens reacted with HTLV-II-specific epitope (gp52env). These results show that recombinant envelope-spiked Western blots provide a simple means for serologic confirmation of STLV-I infection and that type-specific synthetic peptides can be used to confirm the virus type in seropositive monkey specimens.     

Human T lymphotropic virus types I and II proviral sequences in Argentinian blood donors with indeterminate Western blot patterns

Human T-cell lymphotropic virus (HTLV) seroindeterminate blood donors have been reported worldwide including Argentina. To investigate the significance of HTLV-I/II seroindeterminate Western blot (WB) patterns, we conducted an 8-year cross-sectional study. Of 86,238 Argentinian blood donors, 146 sera were reactive by screening tests. The WB results indicated that 20% were HTLV-I reactive, 8% HTLV-II reactive, 61% indeterminate, and 11% negative. The overall seroprevalence was 0.034% for HTLV-I, 0.014% for HTLV-II, and 0.103% for indeterminate. In 57 reactive specimens, HTLV-I/II provirus could be examined by type specific PCR for tax, pol, and env regions. When at least two gene fragments were amplified HTLV-I/II infection was considered confirmed. PCR results confirmed all WB seropositive samples for HTLV-I (n = 15), and HTLV-II (n = 7), and the only WB negative case was also PCR negative, showing a complete concordance between PCR and WB. However, of 34 WB seroindeterminate sera studied by PCR, in 5 was proviral DNA amplified. According to our criteria PCR confirmed one to be HTLV-I, and one HTLV-II, 3 remained indeterminate since only tax sequences were amplified. Among WB indeterminate samples tested by PCR, most of their serological profile showed reactivity to gag codified proteins but lacked env reactivities (70%). One sample with a WB gag pattern showed proviral tax sequences, but of the four samples with reactivity to env proteins GD21 (n = 3) or rgp46II (n = 1) PCR results indicated that one was HTLV-I, one was HTLV-II, and two were indeterminate (only tax sequences). In conclusion, the majority of HTLV-seroindeterminate WB donors exhibited a gag indeterminate profile lacking HTLV provirus, and were thus considered uninfected. However, seroreactivity to env proteins, in particular to GD21, may indicate infection and a follow-up study of each seroreactive blood donor should be considered.     

Spontaneous lymphocyte proliferation in human T-cell lymphotropic virus type I (HTLV-I) and HTLV-II infection: T-cell subset responses and their relationships to the presence of provirus and viral antigen production.

Spontaneous lymphocyte proliferation (SLP) during in vitro culture of mononuclear cells (MCs) characterizes over half of asymptomatic individuals infected with human T-cell lymphotropic virus type I (HTLV-I) or HTLV-II. Both CD4 and CD8 T-cell subsets within MC cultures are activated during SLP, as judged by high-density CD25 (CD25bright) expression; it is unclear, however, whether both cell subsets can directly undergo SLP. In the present investigation, the SLP capacities of purified CD8 and CD4 cells were examined in subjects infected with HTLV-I (n = 19) or HTLV-II (n = 54) in relation to the SLP status of MCs from each subject. No increase in SLP was observed for CD8 or CD4 cells from SLP-negative (SLP-) HTLV-infected subjects, whereas robust SLP characterized CD8 cells from all SLP-positive (SLP+) individuals, regardless of HTLV type. In contrast, SLP+ CD4 cells characterized only 23% (7 of 31) of HTLV-II+ SLP+ individuals, whereas SLP+ CD4 cells characterized 100% of HTLV-I+ SLP+ individuals. In cocultures of HTLV-II+ SLP+ CD8 cells and autologous SLP- CD4 cells, sizable proportions of both CD8 cells and CD4 cells coexpressed CD25bright, suggesting that SLP- CD4 cells were activated in the presence of SLP+ CD8 cells. PCR analysis for tax sequences detected provirus in most CD4- and CD8-cell preparations from HTLV-seropositive individuals, regardless of type and the SLP status of cell subsets. To determine whether SLP was associated with activation of viral genes, levels of HTLV-I and HTLV-II core antigen (Ag) in supernatants were measured. Viral Ag production and SLP responses were significantly correlated for both CD4 and CD8 cells in both HTLV-I and HTLV-II infections. However, inhibition of CD8- or CD4-cell SLP by cyclosporin A or anti-Tac (anti-CD25) did not reduce Ag production, indicating that Ag production is not coupled to SLP. These findings show that CD4 cells from SLP+ HTLV-I+ and SLP+ HTLV-II+ individuals differ in SLP capacity, that the absence of SLP does not indicate a lack of infection, and that production of viral Ag is associated with, but not dependent on, SLP.     

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.     

Development of a supersensitive polymerase chain reaction method for human T lymphotropic virus type II (HTLV-II) and detection of HTLV-II proviral DNA from blood donors in Japan

A supersensitive polymerase chain reaction procedure was developed to detect human T-lymphotropic virus type II (HTLV-II) proviral genome. Six primer pairs covering the various regions of HTLV-II were compared and selected on the basis of specificity and sensitivity. Among them, one primer pair of the pol region of HTLV-II (II pol) was able to amplify and detect even 0.1 fg of the cloned plasmid HTLV-II DNA (seven copies) by regular ethidium bromide staining on polyacrylamide gel. By using this procedure, we screened 189 HTLV-I seropositive blood donors from Yamaguchi and Fukuoka Red Cross Blood Centers, Japan. There were four positive samples detectable with the HTLV-II-specific pol primer pair, as well as with the HTLV-I tax primer pair. The amplified DNAs of two specimens were cloned and sequenced. The sequences of the HTLV-I tax region from both specimens were identical to that of HTLV-I. On the other hand, those of the HTLV-II pol region were identical to that of HTLV-II, except for one base substitution in a clone from one subject. These results indicate that dual infection of HTLV-I and HTLV-II in the same persons occurs among Japanese blood donors.     

Infection with human T lymphotropic virus type I in organ transplant donors and recipients in Spain

Human T-cell lymphotropic virus (HTLV) antibody screening is not recommended uniformly before transplantation in Western countries. In the year 2001, the first cases of HTLV-I infection acquired through organ transplantation from one asymptomatic carrier were reported in Europe. All three organ recipients developed a subacute myelopathy shortly after transplantation. This report rose the question about whether to implement universal anti-HTLV screening of all organ donors or selective screening of donors from endemic areas for HTLV-I infection should be carried out. A national survey was conducted thereafter in which anti-HTLV antibodies were tested in 1,298 organ transplant donors and 493 potential recipients. None was seropositive for HTLV-I and only one recipient, a former intravenous (i.v.) drug user, was found to be infected with HTLV-II. In a different survey, HTLV screening was conducted in 1,079 immigrants and 5 (0.5%) were found to be asymptomatic HTLV-I carriers. All came from endemic areas for HTLV-I infection. No cases of HTLV-II infection were found among immigrants. These results support the current policy of mandatory testing of anti-HTLV antibodies in Spain only among organ transplant donors coming from HTLV-I endemic areas or with a highly suspicion of HTLV-I infection.     

Evaluation of the INNO-LIA HTLV I/II assay for confirmation of human T-cell leukemia virus-reactive sera in blood bank donations

We have evaluated a new serological confirmatory test (INNO-LIA HTLV I/II Ab [INNO-LIA]) for human T-cell leukemia virus (HTLV) using a large collection of samples from Brazilian blood donors (S?o Paulo region) and compared the results with those obtained by Western blotting (WB) tests (WB2.3 and WB2.4). Blood donations were initially screened by enzyme-linked immunosorbent assays (ELISAs) based on viral lysates, and repeatedly reactive samples were further tested by WB2.3. When available, samples were also tested by PCR, two additional ELISAs based on recombinant antigens (recombinant ELISAs), a new-generation WB assay (WB2.4), and the INNO-LIA. Of the 18,169 samples tested, 292 (1.61%) were repeatedly reactive in the ELISAs (viral lysate based) and were further tested by WB2.3; 97 were positive (19 that were typed as HTLV type I [HTLV-I], 12 that were typed as HTLV type II [HTLV-II], and 66 that were nontypeable), 17 were negative, and 178 had indeterminate results. Of the samples with indeterminate results, 172 were tested by INNO-LIA, which could resolve 153 samples as negative. Regarding the positive samples, WB2. 3 and INNO-LIA produced concordant results for all HTLV-I-positive samples, whereas for HTLV-II they agreed for 10 of 12 samples; the 2 samples with discordant results were considered to be positive for HTLV-II by WB with WB2.3 but negative for HTLV-II by INNO-LIA and the two recombinant ELISAs. Furthermore, of the 66 nontypeable samples, 60 underwent testing by INNO-LIA; 54 turned out to be negative by the latter test as well as by recombinant ELISAs. In conclusion, the new serological confirmatory assay for HTLV (INNO-LIA HTLV I/II Ab) resolved the results for the majority of the indeterminate and positive-untypeable samples frequently observed by WB assays.     

Prevalence of infection by human T-cell leukemia virus types I and II in southern Spain

To assess the spread of human T-cell leukemia virus (HTLV) type I and II in different population groups at potential risk of infection in Spain, a total of 756 subjects were studied: 453 belonging to groups at risk for retrovirus infection, 255 with diseases potentially linked to HTLV-I/II infection and 48 immigrants from endemic areas. An HTLV-I viral-lysate enzyme immunoassay (EIA) with a recombinant transmembrane envelope protein incorporated was used to screen serum samples. Reactive specimens were confirmed by Western blot strips spiked with recombinant proteins that differentiated HTLV-I from HTLV-II. Infection was then verified by the polymerase chain reaction (PCR). Serum samples from 19 of the 756 subjects analyzed (2.5 %) were reactive for HTLV by EIA. One of these was from an intravenous drug user (IVDU) in whom HTLV-II infection was confirmed by Western blot and PCR; a specimen from another IVDU showed Western blot reactivity for both retroviruses, but PCR results were negative. Lastly, Western blot confirmed the presence of HTLV in one of the immigrant subjects. Western blot did not verify HTLV infection in the remaining 16 cases, indicating a high rate of nonspecific anti-HTLV reactivity when a second-generation EIA screening test was applied. These results suggest that HTLV is present in Spain among populations at high risk for HTLV, although at a very low rate and restricted to intravenous drug users and individuals immigrating from endemic areas.     

Evaluation of a solid-phase immunoassay for the simultaneous detection of antibodies to human immunodeficiency virus type 1 and human T-cell lymphotropic virus type I.

We describe the evaluation of a solid-phase immunoassay developed for the simultaneous detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) in human serum. The immunoassay employs a mixture of HIV-1 and HTLV-I whole viral lysates immobilized in the wells of microtiter plates. Evaluation of genetically well-pedigreed specimens along with normal blood donor samples indicated that the performance characteristics of the test were equivalent to the sensitivity and specificity of individual tests licensed by the Food and Drug Administration for antibodies to HIV-1 and HTLV-I. Furthermore, the test was also able to detect the presence of cross-reacting antibodies in HTLV-II-infected individuals. The use of such a test would greatly reduce the continually mounting costs associated with screening transfusable products for infectious agents.     

HTLV-I/II infection in a high viral endemic area of Zaire,Central Africa: Comparative evaluation of serology,PCR, and significance of indeterminate Western blot pattern

The frequency of indeterminate Western blot (WB) seroreactivities against HTLV-I “gag encoded proteins” only, and the use of low specific diagnostic WB criteria led to the overestimation of HTLV-I seroprevalence in initial studies in intertropical Africa and Papua New Guinea. In order to clarify the meaning of such seroreactivity, 98 blood samples of individuals from a high HTLV-I endemic area in Zaire, Central Africa were studied by a WB assay containing HTLV-I disrupted virions enriched with a gp 21 recombinant protein and a synthetic peptide from the gp 46 region (MTA-1), and by the polymerase chain reaction (PCR) with 3 primers pairs and 4 different HTLV-I and or HTLV-II-specific probes. These 98 samples were taken mainly from patients with neurological diseases and from their relatives. Using stringent WB criteria, 28 sera (29%) were considered as HTLV-I-positive, 3 as negative and 67 (68%) as indeterminate. A large proportion of these indeterminate sera would have been considered as HTLV-I-positive samples according to previous low specific WB diagnostic criteria. After PCR, 35 samples (36%) were considered as positive for the presence of HTLV-I proviral DNA. Out of the 67 WB seroindeterminate, 10 (15%) were found HTLV-I-positive by PCR. These 10 individuals exhibited in WB multiple band reactivity with p19 and/or p24 (7 cases of both) associated in 6 cases with rgp 21, but never with MTA-1. No samples were found PCR-positive for HTLV-II despite the findings of 11 sera suggestive of HTLV-II by WB. These findings demonstrate that even in a high HTLV-I endemic area, only a minority (about 15%) of the WB-seroinde-terminate individuals could be considered as infected by HTLV-I, and that very stringent WB criteria could lead to overlooking some infected individuals. © 1994 Wiley-Liss, Inc.