Performance of absolute CD4+ count in predicting co-infection with human T-lymphotropic virus type 1 in antiretroviral-naive HIV-infected patients.

Early identification of patients co-infected with HIV and human T-lymphotropic virus type 1 (HTLV-1) is essential to improve care, as CD4+ T-cell counts have been revealed to be an unreliable laboratory parameter to monitor HIV infection in co-infection. Unfortunately, HTLV-1 testing is not currently available in sub-Saharan Africa. We conducted this study to determine the performance of absolute CD4+ T-cell count estimation in guiding the clinical suspicion of co-infection. A cross-sectional survey was conducted in antiretroviral-naïve HIV (AN-HIV) patients attending an HIV outpatient clinic in Maputo city, Mozambique. Seven hundred and one AN-HIV patients were enrolled in the study. The prevalence of HTLV-1 co-infection was 4.5% (95% confidence interval [CI] 3.0-6.0%). Logistic regression analysis showed that CD4+ T-cell count was an independent predictor of co-infection (P value: 0.000). The performance of absolute CD4+ T-cell counts in predicting co-infection was higher in symptomatic HIV patients when compared with asymptomatic HIV patients. The best performance was achieved with the cut-off of CD4+ count of 500 cells/mm(3), which gave sensitivity, specificity, positive and negative predictive values of 54.2%, 87.2%, 24.0% and 96.2%, respectively. In conclusion, our data provide evidence that the absolute CD4+ T-cell count is of moderate accuracy in guiding the clinical suspicion of co-infection in AN-HIV and its implementation could improve the care provided to a significant number of HIV patients in Mozambique.

Evaluation of three methods for hemoglobin measurement in a blood donor setting.

CONTEXT: The hemoglobin (Hb) level is the most-used parameter for screening blood donors for the presence of anemia, one of the most-used methods for measuring Hb levels is based on photometric detection of cyanmetahemoglobin, as an alternative to this technology, HemoCue has developed a photometric method based on the determination of azide metahemoglobin. OBJECTIVE: To evaluate the performance of three methods for hemoglobin (Hb) determination in a blood bank setting. DESIGN: Prospective study utilizing blood samples to compare methods for Hb determination. SETTING: Hemotherapy Service of the Hospital Israelita Albert Einstein, a private institution in the tertiary health care system. SAMPLE: Serial blood samples were collected from 259 individuals during the period from March to June 1996. MAIN MEASUREMENTS: Test performances and their comparisons were assessed by the analysis of coefficients of variation (CV), linear regression and mean differences. RESULTS: The CV for the three methods were: Coulter 0.68%, Cobas 0.82% and HemoCue 0.69%. There was no difference between the mean Hb determination for the three methods (p>0.05). The Coulter and Cobas methods showed the best agreement and the HemoCue method gave a lower Hb determination when compared to both the Coulter and Cobas methods. However, pairs of methods involving the HemoCue seem to have narrower limits of agreement (+/- 0.78 and +/- 1.02) than the Coulter and Cobas combination (+/- 1.13). CONCLUSION: The three methods provide good agreement for hemoglobin determination.

Genetic variation and susceptibilities to protease inhibitors among subtype B and F isolates in Brazil.

The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (prt) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow the gag subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilian prt sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.

Human T-cell leukemia viruses: epidemiology, biology, and pathogenesis.

The human T-cell lymphotropic viruses type I and type II are closely related human retroviruses that have similar biological properties, genetic organization and tropism for T lymphocytes. Along with the simian T-cell lymphoma virus type I, they define the group of retroviruses known as the primate T-cell leukemia/lymphoma viruses. Initially identified in 1980, the human T-cell lymphotropic virus type I has been implicated as the etiologic agent of adult T-cell leukemia/lymphoma and of a degenerative neurologic disorder known as tropical spastic paraparesis or human T-cell lymphotropic virus type I-associated myelopathy. The intriguing link between human T-cell lymphotropic virus type, T-cell malignancy, and a totally unrelated and non-overlapping neurological disorder suggests divergent and unique pathogenetic mechanisms. This review will address the epidemiology, molecular biology, and pathogenesis of human T-cell leukemia viruses.

Chagas' disease: an algorithm for donor screening and positive donor counseling

Classical serological screening assays for Chagas' disease are time consuming and subjective. The objective of the present work is to evaluate the enzyme immuno-assay (ELISA) methodology and to propose an algorithm for blood banks to be applied to Chagas' disease. Seven thousand, nine hundred and ninety nine blood donor samples were screened by both reverse passive hemagglutination (RPHA) and indirect immunofluorescence assay (IFA). Samples reactive on RPHA and/or IFA were submitted to supplementary RPHA, IFA and complement fixation (CFA) tests. This strategy allowed us to create a panel of 60 samples to evaluate the ELISA methodology from 3 different manufacturers. The sensitivity of the screening by IFA and the 3 different ELISA's was 100%. The specificity was better on ELISA methodology. For Chagas disease, ELISA seems to be the best test for blood donor screening, because it showed high sensitivity and specificity, it is not subjective and can be automated. Therefore, it was possible to propose an algorithm to screen samples and confirm donor results at the blood bank.

Os testes sorológicos clássicos utilizados na triagem de doadores de sangue são trabalhosos e subjetivos. O objetivo do presente trabalho é o de avaliar a metodologia imuno-enzimática (ELISA) e propor um algorítmo para doença de Chagas em bancos de sangue. Foram estudados 7999 doadores de sangue e/ou componentes cujas amostras foram testadas com o objetivo de tria-las sorologicamente para doença de Chagas utilizando hemaglutinação passiva reversa (RPHA) e imunofluorescência indireta (IFA). As amostras reativas em pelo menos uma destas metodologias, foram retestadas com reativos diferentes por RPHA, IFA e fixação de complemento (CFA). Esta estratégia nos permitiu criar um painel de 60 amostras com as quais tornou-se possível a avaliação do método imunoenzimático (ELISA). A sensibilidade da triagem dos doadores pelos métodos ELISA e IFA foi de 100%. A especificidade foi melhor para a metodologia imunoenzimática. O teste ELISA parece ser o ideal para triagem em bancos de sangue pois é altamente sensível, específico, não é subjetivo e pode ser automatizado. Desta forma, torna-se possível a formulação de um algorítimo a ser utilizado na triagem sorológica e confirmação de resultados em doadores de bancos de sangue.

Chagas' disease: an algorithm for donor screening and positive donor counseling.

Classical serological screening assays for Chagas' disease are time consuming and subjective. The objective of the present work is to evaluate the enzyme immuno-assay (ELISA) methodology and to propose an algorithm for blood banks to be applied to Chagas' disease. Seven thousand, nine hundred and ninety nine blood donor samples were screened by both reverse passive hemagglutination (RPHA) and indirect immunofluorescence assay (IFA). Samples reactive on RPHA and/or IFA were submitted to supplementary RPHA, IFA and complement fixation (CFA) tests. This strategy allowed us to create a panel of 60 samples to evaluate the ELISA methodology from 3 different manufacturers. The sensitivity of the screening by IFA and the 3 different ELISA's was 100%. The specificity was better on ELISA methodology. For Chagas disease, ELISA seems to be the best test for blood donor screening, because it showed high sensitivity and specificity, it is not subjective and can be automated. Therefore, it was possible to propose an algorithm to screen samples and confirm donor results at the blood bank.

Risk factor analysis and serological diagnosis of HIV-1/HIV-2 infection in a Brazilian blood donor population: validation of the World Health Organization strategy for HIV testing.

OBJECTIVE: To determine the relative prevalence of HIV-1 and HIV-2 and to evaluate the World Health Organization testing strategy for HIV diagnosis in a low-risk population in Brazil. In addition, to assess risk factors for HIV infection. DESIGN: Sera obtained from 9885 consecutive blood donors were screened in parallel by two HIV enzyme-linked immunosorbent assays (ELISA) with different antigen composition and test principles (Ortho HIV-1 and Wellcozyme HIV-1/2). Samples reactive to either ELISA were submitted to a Western blot assay and to a rapid HIV-1/2 ELISA (Sero-Immuno Diagnostics). An ELISA test with specific HIV-1/HIV-2-derived synthetic peptide was used to discriminate between samples reactive on the Wellcozyme HIV-1/2 assay. Demographic and serological data were used to address risk factors for HIV infection. RESULTS: All the 28 Western blot-confirmed positive samples were reactive in both Ortho HIV-1 and Wellcozyme HIV-1/2 assays (sensitivity, 100%). The Wellcozyme HIV-1/2 specificity (99.9%) was higher than Ortho HIV-1 (99.5%). If sample reactivity to both tests was considered positive, the sensitivity and specificity of the screening would be 100%. However, further analysis with a third rapid HIV-1/2 ELISA reduced both the sensitivity and the specificity of the sequential testing strategy. Discrimination between HIV-1 and HIV-2 showed evidence for the presence of HIV-1 only. Finally, in the group aged 18-35 years, the presence of serological markers of hepatitis C virus and hepatitis B virus infections and the elevated levels of beta 2-microglobulin were variables associated with the identification of HIV-1-seropositive blood donors. CONCLUSION: In a low-risk population, application of two high quality ELISA tests with different antigens and test principles can replace the use of the Western blot. In addition to the cost being reduced by 10-16%, a rapid diagnosis and the absence of indeterminate Western blot results confer an advantage to this strategy.

Catalase expression, katG, and MIC of isoniazid for Mycobacterium tuberculosis isolates from São Paulo, Brazil.

The MIC of isoniazid, peroxidase-catalase expression, and the presence of the katG gene for 102 Mycobacterium tuberculosis isolates from patients in São Paulo were compared. Fifty-three isoniazid-resistant and 49 isoniazid-sensitive isolates were analyzed by polymerase chain reaction (PCR) for the presence of katG sequences. All isoniazid-sensitive and 43 (81%) isoniazid-resistant isolates expressed catalase (P = .001). None of isoniazid-sensitive and 4 (7%) of 53 isoniazid-resistant isolates lacked katG sequences. Among 6 isolates with MICs > 50 micrograms/mL, 5 (83%) did not express catalase and 2 lacked katG sequences; only 1 had complete gene deletion shown by Southern blot analysis. These findings indicate a correlation between loss of catalase and isoniazid resistance among highly resistant isolates, but these isolates were a small proportion of resistant clinical M. tuberculosis isolates from São Paulo.

A rapid and reliable assay to enumerate CD4+ T lymphocytes in whole blood.

We compared the performance of a rapid and simple anti-CD4 antibody-coated microsphere assay with flow cytometry and immunofluorescence for quantitation of absolute count of CD4+ T lymphocytes. A longitudinal evaluation of CD4+ T lymphocytes by flow cytometry and microsphere assay in 10 human immunodeficiency virus (HIV)-seronegative and 59 HIV-seropositive individuals was conducted over a period of 9 months. Standard flow cytometry analysis was performed to establish the absolute CD4+ T-lymphocyte count. The microsphere assay uses whole blood; CD14+ and CD4+ cells are first blocked by small latex beads coated with anti-CD14 antibody, and remaining cells are stained with larger anti-CD4 antibody-coated beads. Cells rosetted with only anti-CD4 antibody-coated beads are counted with use of a hemacytometer. Immunofluorescence microscopy was performed by standard techniques with use of peripheral blood mononuclear cells. The predictive value for stratification of HIV-seropositive patients by CD4+ T-lymphocyte values of < 200/microliters was 95% when the microsphere method was compared with flow cytometry. A correlation coefficient of 0.91 between the two assay methods was demonstrated in 281 CD4+ T-lymphocyte tests for absolute count. Finally, the flow cytometry method yielded better results than did the microsphere assay and immunofluorescence microscopy, in descending order of accuracy. The microsphere method should be effective in determining absolute CD4+ T-lymphocyte count in developing countries where, for a variety of reasons, no other method can be reliably performed.

Isolation of Mycobacterium avium complex from bone marrow aspirates of AIDS patients in Brazil.

Mycobacterium avium complex (MAC) infection has not been reported as a major opportunistic infection among patients with AIDS in Latin America or Africa. In this study, 125 AIDS patients who had persistent fever, anemia, and leukopenia were examined among 2628 AIDS patients admitted to Instituto de Infectologia Emilio Ribas between May 1990 and April 1992. From the bone marrow aspirates of the 125 patients, MAC was isolated from 23 (18.4%) and Mycobacterium tuberculosis was isolated from 9 (7.2%). Between 1985 and 1990, only 11 MAC isolations among 60,000 cultures obtained from human immunodeficiency virus-seronegative patients were documented in São Paulo. Hence, the minimal estimated rate of MAC infection in AIDS patients in this city was 23/2628, or 0.88%. These findings suggest that MAC infection is an important opportunistic infection, especially among a subset of patients with AIDS in Brazil who have clinical characteristics and risk activities similar to those associated with MAC infections in North America and Europe.

Phorbol ester-induced differentiation of U937 cells enhances attachment to fibronectin and distinctly modulates the alpha 5 beta 1 and alpha 4 beta 1 fibronectin receptors.

Monocyte interaction with fibronectin (Fn) involves specific cell surface receptors and results in cell attachment and differentiation. We have studied the regulation of these receptors using the promonocytic cell line U937 and its PMA-induced differentiation as a model. We recently reported that U937 cells interact with two sites in Fn, RGD and CS-1, via two independent receptors (O. C. Ferreira, A. Garcia-Pardo, and C. Bianco (1990) J. Exp. Med. 171, 351). In this study we have determined the effects of PMA on the interaction of U937 cells with both sites in Fn. PMA-U937 cells showed an enhanced attachment to Fn and to an RGD-containing 80-kDa Fn fragment. This enhancement paralleled a two- to threefold increase in the surface expression of the RGD-dependent receptor alpha 5 beta 1. An anti-alpha 5 beta 1 mAb completely inhibited cell adhesion to Fn and to the 80-kDa fragment. alpha 5 beta 1 receptors from untreated and PMA-treated U937 cells were isolated on 80-kDa-Sepharose columns and shown to contain a similar complex of 152/125-kDa proteins, although proteins from PMA-treated cells had slightly faster mobility on SDS-gels. In contrast, the total number of PMA-U937 cells adhering to a 38-kDa Fn fragment (containing the CS-1 site) was lower when compared to that of untreated cells. This decrease was accompanied by a 50% loss of cell surface alpha 4 beta 1, the specific receptor for CS-1. Our results indicate that differentiation of U937 cells enhances adhesion to Fn primarily by up-regulating the alpha 5 beta 1 Fn receptor. PMA also induces a down-regulation of alpha 4 beta 1, suggesting that these two integrins play different roles during monocyte differentiation.

Human B lymphocytes define an alternative mechanism of adhesion to fibronectin. The interaction of the alpha 4 beta 1 integrin with the LHGPEILDVPST sequence of the type III connecting segment is sufficient to promote cell attachment.

In this report we have studied the mechanism of human B lymphocyte adhesion to fibronectin and to proteolytic fragments of this protein. B cells adhered to fibronectin and to a 38-kDa fragment, derived from the A chain, containing the Hep II domain and most of the type III connecting segment, IIICS, of fibronectin. Cells did not bind to an 80-kDa fragment containing the RGD adhesive sequence of fibronectin. Attachment to fibronectin or to the 38-kDa fragment was not affected by the 80-kDa fragment, the GRGDSPC synthetic peptide, or by a mAb specific for the alpha chain of the RGD-dependent fibronectin receptor, alpha 5 beta 1. However, B cell adhesion to fibronectin was inhibited by the synthetic peptides CS-1, comprising the first 25 amino acids of IIICS and B12, containing the sequence LHGPEILDVPST of CS-1 (residues 14-25). Moreover, this sequence was shown to be sufficient to induce stable cell adhesion when coated on plastic surfaces. A mAb specific for the alpha-subunit of the alpha 4 beta 1 integrin, completely inhibited B cell attachment to B12, CS-1, 38 kDa, and fibronectin coated substrata. These data clearly indicate that adhesion of B lymphocytes to fibronectin is exclusively mediated by the interaction of alpha 4 beta 1 with residues 14-25 of the IIICS region in fibronectin. Therefore this interaction constitutes an alternative pathway of adhesion to fibronectin, independent of RGD and alpha 5 beta 1.

Specific binding of the human monocytic cell line U937 to the alternatively spliced connecting segment (IIICS) of fibronectin.

U937 cells attach to the RGDS-containing 80-kD fragment of fibronectin (Fn). The present report examined whether these cells recognize other domains of Fn. U937 cells attach to a 38-kD fragment derived from the A chain of Fn, which includes the Hep II domain and most of the alternatively spliced IIICS region. U937 did not bind to a 58-kD fragment derived from the B chain (which lacks IIICS) and has the Hep II site. They also did not bind to a 31-kD COOH-terminal fibrin-binding fragment or to a 29-kD fragment containing the Hep I domain. Cell adhesion to the 38-kD fragment was not inhibited by the 80-kD fragment, by GRGDSPC synthetic peptides, or by a mAb directed to the RGDS-containing domain of Fn. Attachment was completely inhibited by the 38-kD fragment and by the synthetic peptide CS-1, comprising the first 25 amino acid residues of IIICS. These results indicate that U937 cells interact with two sites of Fn, the RGDS-containing region, and the IIICS region.

Adhesion of human T-lymphoid cells to fibronectin is mediated by two different fibronectin domains.

Lymphocyte adhesion to components of extracellular matrices (i.e. fibronectin) is important for their proper localization in tissues and inflammatory sites. We have studied the attachment of the human cell line HUT-78 (mature T lymphocytes) to fibronectin and to several tryptic fragments of fibronectin. HUT-78 cells effectively adhered to surfaces coated with two Hep II domain-containing fragments of 38,000 and 58,000 MW derived from the A and B chains of fibronectin, respectively. Cells also bound to an 80,000 MW fragment containing the RGDS sequence of fibronectin. Cell adhesion to the 38,000 MW fragment was completely inhibited (100%) by cell preincubation with the soluble 38,000 MW fragment; it was partially inhibited (30-37%) by preincubation with the 58,000 MW fragment or with a synthetic peptide CS-1, comprising the first 25 amino acid residues of the alternatively spliced connecting segment (IIICS), which is present in the A chain of fibronectin and in the 38,000 MW fragment. Cell preincubation with RGDS-containing synthetic peptides or with the 80,000 MW fragment, did not affect attachment to 38,000 MW-coated surfaces. Moreover, preincubation of HUT-78 cells with 38,000 MW fragment had no effect on cell adhesion to 80,000 MW-coated wells, while preincubation with 80,000 MW fragment completely inhibited cell attachment to these surfaces. These results strongly suggest the involvement of two different cell surface receptors which recognize the Hep II/IIICS site and the RGDS site independently. Preincubation with either 38,000 or 80,000 MW fragments prevented cell attachment to fibronectin, indicating that adhesion to the intact molecule requires interaction with both regions. Therefore T-lymphocyte adherence to fibronectin-containing matrices may be regulated by the co-expression of both receptors at the cell surface.

Fibronectin receptors of mononuclear phagocytes: binding characteristics and biochemical isolation.

Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, we have investigated the hypothesis that proteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. We report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. The Ki for inhibition of attachment (0.5 microM) was consistent with the Kd for binding of the 3H-labeled 80-kDa fragment (0.34 microM) to U937 cells in suspension. There were 4-5 x 10(5) 80-kDa binding sites per cell. The relatively high affinity of the 80-kDa fragment for the monocyte surface permitted the isolation and characterization of fibronectin-binding proteins from U937 cells and peripheral blood monocytes by affinity chromatography. When octylglucoside lysates of lactoperoxidase iodinated cells were applied to 80-kDa-Sepharose columns, a polypeptide complex of 152/125 kDa was eluted with the synthetic peptide GRGDSPC, but not with GRGESP. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.

Autologous induction of the human T-cell-dependent monocyte procoagulant activity: a possible link between immunoregulatory phenomena and blood coagulation.

Activated monocytes acquire the ability to induce clot formation in platelet-poor citrated human plasma. The generation of this procoagulant activity (PCA) is dependent upon an interactive pathway between monocytes and T lymphocytes. Here we show that an ongoing autologous mixed lymphocyte reaction (AMLR) can elicit a T-cell-instructed PCA. PCA was measured by the ability of the cells to accelerate the clotting time of pooled citrated platelet-poor human plasma. AMLR was measured by tritiated thymidine incorporation. PCA and AMLR had very similar kinetics. Correlation coefficients between both reactions ranged from 0.59 to 0.99. Addition of an anti-DR monoclonal antibody blocked both reactions. T-Lymphocyte-depleted cell populations did not increase their level of PCA after 6 days in culture. Addition of autologous T cells to the T-depleted population restored its ability to produce PCA. Cyclosporin A blocked the peripheral blood mononuclear cell ability to generate PCA. A lymphokine generated during the AMLR was able to induce PCA in normal mononuclear cells. The results indicate that self recognition activates monocytes to produce PCA and suggests that this mechanism may represent a link between immunoregulatory phenomena and blood coagulation.

Monocyte procoagulant activity induced by concanavalin A is decreased in a population of chronic chagasic patients.

1. The ability of peripheral blood mononuclear cells (PBMC) from Chagas' disease patients to induce monocyte procoagulant activity (PCA) in response to concanavalin A (Con A) and to a Con A induced lymphokine was studied. 2. In spite of the variability of PCA levels among both chagasic patients and normal controls, statistical analysis of the data permitted us to draw the following general conclusions. 3. The Con A-induced monocyte PCA measured in 17 assays of 15 chagasic patients was significantly lower than the PCA response of 18 normal controls or of 11 patients with non-chagasic myocardiopathies. 4. The response of PBMC from chagasic patients to lymphokine was also lower than that observed for normal controls. 5. These results suggest that monocytes from chagasic patients are deficient for the generation of PCA which is an in vitro correlate of the delayed-type hypersensitivity reaction.