Human herpesvirus-8 infection in hemodialysis patients from São Paulo, Brazil: preliminary results.

Epidemiological studies conducted in endemic areas of human herpesvirus 8 (HHV-8) infection have shown iatrogenic Kaposi's sarcoma in renal transplant recipients. Hemodialysis has not yet been demonstrated to be a route of virus transmission/acquisition, although recently blood transfusion has been suggested as a vehicle of HHV-8 transmission. The present study searching HHV-8 antibodies among serum samples from 70 hemodialysis patients disclosed a high prevalence of infection (22.9%). There was an association between HHV-8 seroreactivity and previous transfusions and transplantation, as well as with a black/pardum ethnic background of patients. These results emphasized that chronic renal patients are at risk of developing HHV-8-related diseases.

Increasing seroprevalence of human herpesvirus 8 (HHV-8) with age confirms HHV-8 endemicity in Amazon Amerindians from Brazil.

Human herpesvirus 8 (HHV-8) seroprevalences were determined in two isolated Amazon Amerindian tribes, according to age, gender and familial aggregation. Plasma and serum samples obtained from 982 Amazon Amerindians (664 Tiriyó and 318 Waiampi) were tested for antibodies against lytic and latent HHV-8 antigens by using 'in-house' immunofluorescence assays. Overall, HHV-8 seroprevalence was 56.8 % (57.4 % in the Tiriyó tribe and 55.7 % in the Waiampi tribe). Seroprevalence was independent of gender and increased linearly with age: it was 35.0 % among children aged 2-9 years, 51.4 % in adolescents (10-19 years), 72.9 % in adults and 82.3 % in adults aged >50 years. Interestingly, 44.4 % of children under 2 years of age were HHV-8-seropositive. No significant differences in seroprevalence between tribes and age groups were detected. It is concluded that HHV-8 is hyperendemic in Brazilian Amazon Amerindians, with vertical and horizontal transmission during childhood, familial transmission and sexual contact in adulthood contributing to this high prevalence in these isolated populations.

Detection of the 20-kDa virulence-associated antigen of Rhodococcus equi in malakoplakia-like lesion in pleural tissue obtained from an AIDS patient.

A malakoplakia-like lesion was detected in a pleural biopsy from an AIDS patient presenting clinical and radiologic features of pneumonia. Cultures of bronchoalveolar lavage and pleural fluid evidenced Rhodococcus equi as the causative agent of pleuro-pulmonary infection. Immunochemical characterization of the R. equi isolate showed the presence of a strain similar to the ATCC 33704 reference strain presenting the capsular antigen of serotype 4, and the intermediate virulence-associated antigen of 20-kDa. Histopathology of the patient's pleural biopsy showed plaques of macrophages interspersed with lymphocytes, and intracytoplasmic cocci and bacilli in macrophages, which were variably acid-fast positive. Immunohistochemistry of cocci, bacilli and their degradation products resulted strongly positive when stained with a mouse monoclonal antibody (MAb) produced against the 20-kDa antigen. This finding could have important implications for the pathogenicity of R. equi for human beings, since we do not know yet all the factors involved in the formation of malakoplakia. Indeed, the results obtained in the present study, taken together with the results obtained for pigs inoculated with R. equi strains of intermediate virulence (Madarame et al. 1998), raise the possibility that most strains presenting the 20-kDa antigen may be capable of inducing malakoplakia. If this hypothesis is confirmed by immunohistochemical analysis of human pulmonary malakoplakia cases due to R. equi, the detection of this antigen may be extremely helpful in the diagnosis and treatment of such patients. This is the first report of R. equi infection in human beings that suggests a relationship between pleural malakoplakia and the virulence-associated antigen of 20-kDa.

HTLV-I/HTLV-II coinfection in an AIDS patient from São Paulo, Brazil.

A serological survey for HTLV infection identified an AIDS patient with HTLV-I/HTLV-II dual seroreactivity. Two further sequential blood samples were collected (samples A and B) for PCR, restriction fragment length polymorphism (RFLP), and sequence analyses of HTLV-I and HTLV-II strains. PCR analyses confirmed dual infection in both samples. Restriction digests of the env region amplified from sample A showed the presence of an HTLV-IIa subtype; the HTLV-II provirus was found to be defective in the pol and env regions in the second sample from this patient. RFLP analysis of the HTLV-II LTR region of both samples confirmed this finding and identified an a5/bzl restriction type. Nucleotide sequence analyses revealed full homology in the HTLV-I env and LTR regions and in the HTLV-II LTR region between the two samples. These findings document the first case of an HTLV-I/HTLV-II coinfection that was fully confirmed and characterized by means of molecular analyses.

Search for an antibody profile of Rhodococcus equi infection in AIDS patients despite the diversity of isolates and patient immune dysfunction.

Diversity of virulence-associated antigens of Rhodococcus equi was detected among thirteen strains isolated from AIDS patients on two continents. One out of four Brazilian isolates presented the virulence-associated antigen of 15- to 17-kDa, and the other three isolates had the 20-kDa virulence-associated antigen. In contrast, only three out of nine Italian isolates were positive for virulence-associated antigens - two for the 15- to 17-kDa antigen and one for the 20-kDa antigen. In four other Italian strains, one or more other low-molecular-weight antigens were identified. Because of R. equi variability and host immune dysfunction, no characteristic antibody profile was detected among patients, although the presence of specific antibodies in serum samples suggested prognostic value: good patient outcome and recovery from pneumonia were correlated with R. equi antibody detection, whereas the lack or disappearance of specific antibodies, mainly those to low-molecular-weight antigens, was correlated with disease progression and patient death. These results confirmed the nonobligatory presence of the well-known virulence-associated antigens for the pathogenicity of R. equi in humans, and also the diversity of R. equi strains isolated from AIDS patients, which may be related to the geographic origin of the isolates or may be a consequence of the route of R. equi transmission in different countries. Some mechanisms underlying the results obtained are discussed, suggesting immune complex formation during the progress of the disease.

Use of V3 loop peptide-specific antibody evaluation for subtyping HIV-1: results of a vertical transmission study from São Paulo, Brazil.

Plasma samples obtained from 97 children enrolled in a longitudinal study of HIV-1 perinatal transmission in São Paulo, Brazil, were tested for the presence of specific V3-loop antibodies in order to determine the HIV-1 subtype circulating among them. A set of seven synthetic peptides representative of the predominant HIV-1 subtypes detected in Brazil was employed in an in-house enzyme immunoassay (EIA) using two different protocols, one of which permits identification of high avidity antibodies (HAAb). Using these approaches we were able to detect antibodies in 64 out of 97 children, independently of the HIV-1 infection status, indicating the presence of subtype B in all cases, except one, which could be considered to be of subtype F. Among subtype B cases, half of the samples reacted with the GWGR motif (type W is representative of Brazilian B strains). In the main, concordant results were obtained between peptide-EIA and HIV-1 status among infants, although in several cases of truly HIV-1 infected children, negative results could be observed. Thirteen mother-child pairs and four fathers were also evaluated, and the results confirmed subtype B to be the prevalent one among them, showing similar proportions of P and W types. Taken together, the results obtained identified subtype B (W and P) uniformly among adults and HIV-1 infected children from São Paulo, Brazil, and confirm vertical and sexual transmission of the predominant strains.

No evidence of vertical transmission of HTLV-I and HTLV-II in children at high risk for HIV-1 infection from São Paulo, Brazil.

One hundred and seven plasma specimens obtained from children born to HIV-1 infected mothers were tested for the presence of antibody to human T-cell lymphotropic virus types I and II (HTLV-I and -II) to determine perinatal transmission of these agents. None of the children in this study were breastfed. Fifty-five specimens were from HIV-1 infected children, 28 from HIV-1 non-infected children, and in 24 cases the HIV-1 status could not be defined. In these series, when ELISA screening tests were employed, HTLV antibodies were detected in 54.5, 17.9, and 37.5 per cent of cases, respectively, given an overall ratio of 41.1 per cent. Western blot analysis disclosed 17 specimens with some HTLV reactivity: three were classified as HTLV-I/II, two confirmed as having a HTLV-I Western blot profile, and the last 12 samples showed reactivity to only one of the protein (gag or env) components. In 11 out of 17 cases molecular approaches were used to confirm HTLV infection in children; no case of HTLV-I or -II was detected. In contrast, when 13 specimens of mother-child pairs were analysed, three mothers' plasma samples which were seropositive were confirmed to have HTLV infection by PCR analysis; one case of HTLV-I and two cases of HTLV-II infections were detected. Taking into account the age of the children and their Western blot profiles, passive maternal antibodies could be detected until the age of 15 months. Indeed, after the age of 18 months seroreactivity amongst the children, with ELISA and Western blot assays, suggests the presence of maternal antibodies that resist degradation and/or antibodies that cross-react with rgp21 or p19 antigens from HTLV, or alternatively, with components of the HIV-1. These results emphasize the lack of HTLV-I and -II vertical transmission in children at high risk who are not breastfed.

Search for Virulence-Associated Antigens of Rhodococcus equi in Strains Isolated from Patients with Acquired Immunodeficiency Syndrome.

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

Sensitivity of two enzyme-linked immunosorbent assay tests in relation to western blot in detecting human T-cell lymphotropic virus types I and II infection among HIV-1 infected patients from São Paulo, Brazil.

We investigated the presence of human T-cell lymphotropic virus types I and II (HTLV-I and HTLV-II) infections, first searching for specific antibodies in 553 serum samples obtained from HIV-1-infected patients from São Paulo, Brazil. Sera were screened using two enzyme-linked immunosorbent assays (ELISAs): the ELISA-EM (ELISA HTLV-I/II, EMBRABIO, BR), which contains HTLV-I and HTLV-II lysates, and the ELISA-DB [ELISA HTLV-I/II, Diagnostic Biotechnology (DB), Singapore], which contains HTLV-I lysate, and HTLV-I and HTLV-II recombinant env proteins (MTA-1 and K55, respectively). Serum samples showing two positive and/or borderline results were confirmed by Western blot (WB 2.3, DB), which discriminates HTLV-I from HTLV-II. WB analyses disclosed 22 cases (4.0%) of HTLV-I and 34 (6.1%) of HTLV-II seroreactivity; 24 sera had indeterminate antibody profile (4.3%) and 2 specimens showed reactivity to both MTA-1 and K55 env proteins. Using stringent WB criteria and analyzing the population according to risk factors, the prevalence rates of HTLV-I and HTLV-II infections were 11.2% and 16.8% in i.v. drug users, 3.4% and 5.5% in heterosexual individuals, and 1.4% and 2.2% in homosexual/bisexual men, respectively. A comparison of ELISA and WB results disclosed that both ELISAs were highly sensitive in detecting HTLV-I antibodies, whereas the ELISA-DB showed 82% sensitivity and the ELISA-EM 100% sensitivity in detecting HTLV-II antibodies. PCR analyses conducted on 37 representative cells samples confirmed the presence of HTLV proviral DNA in the majority of concordant serological cases, except in one, which was HTLV-I infected and seroreacted with K55 protein of HTLV-II. Indeed, after PCR, one case of HTLV-I infection and HTLV-II coinfection, and 30% of WB-seroindeterminate or inconclusive cases infected with HTLV-II could be detected. Our data stress high prevalences of both HTLV-I and HTLV-II infections in HIV-1 coinfected i.v. drug users from São Paulo, and suggests that ELISA kits containing only K55 protein as the HTLV-II-specific antigen, may not have the appropriate sensitivity for the detection of HTLV-II infection in this geographic region, pointing out the need of improved screening tests to be used in Brazil.

Prevalence of HTLV-I and HTLV-II infections among HIV-1-infected asymptomatic individuals in São Paulo, Brazil.

Human immunodeficiency virus (HIV-1)-infected subjects with acquired immunodeficiency syndrome (AIDS) are often infected with multiple pathogens. In particular, HTLV-I and HTLV-II infections have been found more frequently in AIDS patients than in asymptomatic individuals in Europe and Japan. We carried out a serosurvey among asymptomatic HIV-1-infected subjects in São Paulo, Brazil and compared our results with those of other investigators. In this study, we found HTLV infection in 1.5% of 266 asymptomatic and 14% of 28 AIDS patients. Epidemiological data obtained from patients pointed out the use of intravenous drugs as the principal risk factor for acquiring retroviruses. In conclusion, our results are in accordance with other studies done in Brazil and elsewhere where the principal risk group for HIV/HTLV-I/II coinfection was IDU.

Use of Rhodococcus equi virulence-associated protein for immunization of foals against R equi pneumonia.

OBJECTIVE: To evaluate use of the virulence-associated protein of Rhodococcus equi in immunizing foals against R equi pneumonia. ANIMALS: Eight (experimental group) and 6 (controls) mares with their foals. PROCEDURE: Virulence-associated protein extracted from R equi was used to prepare an acetone-precipitated. Triton X-extracted (APTX) antigen. After determination of the efficacy of passive immunization, in untreated foals or in foals given plasma from a horse vaccinated with APTX antigen or from a nonvaccinated horse, a field trial was done to evaluate the efficacy of vaccination of 8 mares, twice with APTX before parturition, and of their foals at ages 3 and 5 weeks; 6 mares and their foals served as unvaccinated controls. All 2-day-old foals were given plasma from local donor horses inoculated with a locally produced bacterin. Serum opsonizing activity produced by vaccination with APTX was determined. Passively immunized foals were challenge exposed with an aerosol of virulent R equi. Foals of the field trial were exposed to enzootic R equi infection. RESULTS: Inoculation with APTX resulted in high IgG antibody liters with opsonizing activity. Passive immunization of foals with plasma from an immunized horse enhanced bacterial clearance from the lungs, compared with that in foals not given plasma or given plasma without APTX antibodies. Vaccination of mares and foals exposed to natural infection resulted in development of R equi pneumonia in 4 of 8 vaccinated foals, but in only 1 of 6 unvaccinated foals. CONCLUSIONS: Vaccination with APTX antigen led to high-titer, opsonizing antibody. Plasma from a vaccinated horse appeared to enhance clearance of R equi from the lungs of foals. Paradoxically, vaccination of mares and their foals with APTX antigen did not protect foals and may have enhanced R equi pneumonia in the foals.

Detection of Herpes Virus (KSHV) DNA Sequences in Brazilian Patients With AIDS-Associated Kaposi's sarcoma.

To explore the possible involvement of herpes virus (KSHV) in AIDS-associated Kaposi's sarcoma (KS) in 7 patients in Brazil, we analyzed 7 AIDS-KS lesions. Using PCR, we found KSHV specific sequences in 3 cases and by using nested PCR, we identified sequences in each of the 7 cases. Direct sequencing on nested-PCR products showed a certain degree of variability in relation to classic KSHV sequences, and identified alterations similar to those described in some endemic cases from Africa and in AIDS-associated KS specimens from North America. This mixed pattern of KSHV sequences observed in AIDS-associated KS from Brazil may reflect the geographic origin of the samples, consistent with the environmental and epidemiological backgrounds of people in this country. It is apparent that, just as in other countries in the world, Kaposi's sarcoma in HIV patients is related to herpes virus infection.

The CD4+ T-cell network and the cytokine profile after HIV-1 infection.

The present article discusses CD4+ T-cell interaction and cytokine production after HIV-1 infection and progression to AIDS. On the basis of the experience of the author with biological fluids obtained from patients suspected of having AIDS and the recently available data concerning this matter, a model is proposed for the CD4+ T-cells network and CD4+ T-cells destruction during this infection. The mechanism of cellular killing involves apoptosis and preferential destruction of activated TH0/TH2-type cells. This type of cells is generated as an immune response to HIV-1 itself or to allergens and helminth infestations. The virus replicates more effectively in activated TH0/TH2-type cells and this contributes to the development of full-blown AIDS. The author has previously proposed the hypothesis of an elevation in IL-5 production during later stages of the disease and the use of eosinophilia of unknown etiology as a prognostic marker of AIDS in developing countries (Caterino-de-Araujo (1994). Immunology Today, 15: 498-499). At the present time, this proposition is confirmed and the use of eosinophilia as an indicator of a shift to a TH0/TH2-type response that predicts progression to AIDS is justified.

[Difficulties in diagnosing atypical primary HIV-1 infection: report of a case]. / Dificuldades no diagnóstico de infecção primária atípica por HIV-1. Relato de um caso.

Several cases of primary HIV-1 infection are not identified, either because the diagnosis is not suspected or because they test negative for HIV-1 antibody. This work presents an uncommon case of primary HIV-1 infection in an young parenteral drug abuser man, who presented symptoms of acute hepatitis. During the initial acute phase the serum sample of the patient tested negative for the presence of antibodies against several viruses, including HIV-1. Nevertheless, the diagnosis of primary HIV-1 infection was suspected by using an alternative method for "in vitro" induced antibody production (IVIAP), and confirmed by p24 antigen serum positivity and seroconversion in serial plasma samples of the patient. The authors suggest the use of the IVIAP and others complementary assays to help the diagnosis of acute HIV-1 infection in persons at high risk conditions.

Rapid in vitro detection of HIV-1-specific antibody secretion by cells-culture with virus antigens.

The present report describes an alternative method for in vitro detection of HIV-1-specific antibody secretion in 24h of culture employing as stimulant of peripheral blood mononuclear cells the disrupted inactivated whole virus adsorbed onto microwells in a commercial ELISA kit plates. The results obtained from this technique have showed high sensitivity and specificity since it was capable of detecting HIV-1 infection early after birth. There were neither false-positivity nor false-negativity when blood samples obtained from HIV-1 seronegative asymptomatic individuals, and HIV-1 seropositive adult patients were analyzed. This rapid, low cost, simple, highly sensitive and specific assay can be extremely useful for early diagnosis of pediatric HIV infection.

An alternative method for in vitro production of HIV-1-specific antibodies.

We describe the detection of HIV-1-specific antibody secretion by a 24-h culture of peripheral blood mononuclear cells (PBMC) stimulated with disrupted inactivated whole virus adsorbed onto commercial ELISA microwell plates. The method showed high sensitivity and specificity and was capable of accurately detecting HIV-1 infection early after birth (9 of the 19 patients were less than 3 years old) because maternal antibodies do not interfere by giving false-positive results. No false-positive results were obtained with PBMC from 24 HIV-1-seronegative asymptomatic individuals and no false-negative results were obtained for 10 seropositive adult patients (16-46 years of age). This rapid, relatively low cost, simple, highly sensitive and specific assay can be extremely useful for the early diagnosis of pediatric AIDS cases.