Human Herpesvirus type 8 (HHV-8): study of target segments of the HHV-8 genoma in blood, saliva and urine of HIV/aids infected patients with or without Kaposi"s Sarcoma / Herpesvírus humano tipo 8 (HHV-8): estudo de segmentos alvo do genoma viral em amostras de sangue, saliva e urina de pacientes infectados pelo HIV/aids, com e sem Sarcoma de Kaposi

Desde a descoberta do herpes vírus humano tipo 8 (HHV-8) como o agente etiológico do sarcoma de Kaposi (SK) nas suas diferentes formas clínico-epidemiológicas, vários estudos vêm sendo conduzidos com o intuito de determinar as vias de transmissão desse vírus em populações endêmicas e de risco epidemiológico. Em regiões endêmicas, a transmissão viral foi relacionada à transmissão horizontal de mães para filhos e entre irmãos e a sexual principalmente, nos casos de SK/aids. Com o objetivo de determinar segmentos do genoma viral em fluídos biológicos e consequentemente seu potencial infectante foi conduzido o presente trabalho. Foram avaliados quanto à presença de segmentos localizados em posições estratégicas do genoma do HHV-8 em sangue, saliva e urina de 76 pacientes com SK/aids, 19 pacientes com HIV/aids, 4 casos de SK clássico e 11 indivíduos sadios (HIV-soronegativos, sem SK). Foram utilizadas as técnicas de PCR "nested" para as ORF K1, ORF 25, ORF 26, ORF K8.1 e ORF 73 em DNA extraído de material de biópsia de lesão de SK (controle positivo), células do sangue periférico, saliva e urina. Os resultados de PCR positivo para o HHV-8 foram analisados quanto a variáveis epidemiológicas, clínicas e laboratoriais. Foram consideradas como variáveis: sexo, cor, origem étnica, tempo de infecção por HIV e de acompanhamento do SK, terapia ARV e para SK, contagem de células CD4+ e sorol

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.

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.

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.

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.

Seropositivity to Chlamydia trachomatis in prostitutes: relationship to other sexually transmitted diseases (STDs).

The prevalence of Chlamydia trachomatis and its relationship with other sexually transmitted diseases (STDs) was investigated by serological determinations in a group of 45 women working as prostitutes in Santos, State of São Paulo. Seropositivity to HIV-1 was demonstrated in 4 (9%) of the cases and to HIV-2 in one case. Syphilis and hepatitis B were detected in 29% and 43% of the 45 women, respectively. Specific antibodies to C. trachomatis were found in all subjects. The high seroprevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), syphilis and C. trachomatis in this population was related to predisposing factors such as number of sexual contacts, sexual practices, drug use and episodes of sexually transmitted diseases (STDs).

PIP: Researchers enrolled 600 prostitutes from an AIDS control and prevention program in a study to determine the prevalence of Chlamydia trachomatis in prostitutes and other sexually transmitted diseases (STDs). The prostitutes worked in the port city of Santos, Brazil where many people use intravenous (IV) drugs. Only 45 prostitutes met the study criterion of 5-100 sexual partners/day. Health practitioners took sera from each woman to test for HIV-1, HIV-2, hepatitis B surface antigen (HBsAg) and antibody (HBsAb), Treponema species (syphilis), and C. trachomatis. All the women tested positive for C. trachomatis. This high percentage may have been due to previous contact with the microbe and not necessarily due to an active infection. 42% had been exposed to Treponema. 20% were HBsAb seropositive and 9% HBsAg seropositive. 9% tested positive for HIV-1 and 2% for HIV-2. In another study in Campinas, Brazil, HIV-1 and seropositivity was 21.5% for prostitutes and transvestites. In addition, in a study in metropolitan Sao Paulo, HIV infection prevalence varied from 18-73% among 935 women and 22% among prostitutes. 58% of the prostitutes in Santos had had sexual intercourse with bisexuals or IV drug users. 44% had previously experienced an STD. 42% used IV drugs. 42% practiced both oral and vaginal sex. 36% practiced oral, vaginal, and anal sex. Only 22% limited themselves to oral sex. Since C. trachomatis can cause infertility, chronic pelvic pain, and spontaneous abortion and since every prostitute in the study had been exposed to it, health workers should institute regular STD screening for prostitutes.

Neutropenia in an infant: possible autoimmune etiology.

The present study was undertaken to determine if the serum of a child with severe neutropenia contained antibodies against parental neutrophils. The presence of IgG antibodies to granulocytes from both parents was demonstrated using the indirect immunofluorescence technique. These data suggest an autoimmune etiology for the neutropenia of this patient.

Primary immunodeficiency diseases: a presentation of 6 cases.

The peripheral blood leukocytes of 6 children with clinical data suggestive of primary cellular immunodeficiencies were studied in an attempt to define the cellular basis of these disorders. The phenotype and function of T and B cells were investigated. According to the clinical and laboratory features, the patients were classified as one case of severe combined immunodeficiency (SCID), two of ataxia-telangiectasia (AT), one of Wiskott-Aldrich syndrome (WAS), one of DiGeorge syndrome (DSG), and one of cellular immunodeficiency (CID). The laboratory investigations together with the clinical manifestations permitted a diagnosis of primary immunodeficiency diseases.