HTLV-I/II and blood donors: determinants associated with seropositivity in a low risk population

OBJECTIVE: Blood donors in Brazil have been routinely screened for HTLV-I/II since 1993. A study was performed to estimate the prevalence of HTLV-I/II infection in a low risk population and to better understand determinants associated with seropositivity. METHODS: HTLV-I/II seropositive (n=135), indeterminate (n=167) and seronegative blood donors (n=116) were enrolled in an open prevalence prospective cohort study. A cross-sectional epidemiological study of positive, indeterminate and seronegative HTLV-I/II subjects was conducted to assess behavioral and environmental risk factors for seropositivity. HTLV-I/II serological status was confirmed using enzyme-linked immunosorbent assay (EIA) and Western blot (WB). RESULTS: The three groups were not homogeneous. HTLV-I/II seropositivity was associated to past blood transfusion and years of schooling, a marker of socioeconomic status, and use of non-intravenous illegal drugs. CONCLUSIONS: The study results reinforce the importance of continuous monitoring and improvement of blood donor selection process.

Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

Diminuição do risco de infecção pelo vírus da imunodeficiência humana (VIH) em pacientes em hemodiálise no estado do Rio de Janeiro, Brasil

Os estudos iniciais sobre a soroprevalência de anticorpos anti-VIH-1 (Ac-VIH) em unidades de hemodiálise no Estado do Rio de Janeiro (RJ) foram feitos em 1985. Os números alarmantes, próximos a 14%, foram atribuídos à má qualidade do sangue obtido de "doadores profissionais" em troca de comida ou dinheiro. Recentemente uma série de medidas foram adotadas na tentativa de reduzir o tráfico de sangue. Nossa investigação objetivou avaliar o impacto destas na soroprevalência de Ac-VIH em duas unidades satélites no RJ. A Clínica Segumed foi uma das unidades estudadas em 1985. Em 1987 realizamos um segundo levantamento no mesmo grupo estudado previamente. A Casa de Saúde Grajaú, inaugurada em 1986 com a maioria dos pacientes novos em diálise, foi estudada em 1988. O teste ELISA HIV-1 foi utilizado como rastreamento. Os resultados positivos foram confirmados com Western blot. Os resultados na Segumed mostraram uma grande diferença entre os dois levantamentos (14,4% vs 3,6%). Os dois casos positivos em 1987 estavam entre os identificados em 1985. Nenhum paciente se infectou entre os dois levantamentos apesar de não se utilizarem medidas de isolamento para os portadores de VIH e do uso de transfusões ter aumentado no período. Na CS Grajaú apenas dois casos foram encontrados (soroprevalência 2,4%) embora um já fosse conhecido desde 1985 quando vivia com um transplante. Uma revisão de estudos semelhantes no RJ e São Paulo parece revelar uma tendência à diminuição das taxas nos últimos anos. Nós concluímos que a chance de contaminação com VIH é atualmente reduzida nos centros estudados e pode estar caindo globalmente no RJ. É possível que a maior vigilância, e até fechamento de bancos de sangue, tenha resultado na melhora da qualidade do sangue no RJ.

Antibody to human T-lymphotropic virus in a patient with Guillain-Barré syndrome (case report)

Serum sample obtained from a male, 12 year old patient suffering from Guillain-Barré syndrome (GBS) was positive for human T-lymphotropic virus (HTLV-I) antibody by the enzyme-linked immunosorbent assay (ELISA) and the Western Blot analysis (WB). Attempts to isolate enteroviruses (including poliovirus) from faecal material in both tissue culture and suckling mice were unsuccessful; in addition, acute and convalescent paired serum samples did not show any evidence of recent poliovirus infection when tested against the three serotypes. Specific tests for detection of Epstein-Barr virus infection were not performed; however, the Paul-Bunnel test yielded negative results. ELISA for detection of anti-cytomegalovirus IgM was also negative. The concomitant occurrence of either adult T cell leukemia (ATL) or lymphoma was not recorded in this case.

Serological, electrophoretic and biological properties of Fasciola hepatica antigens

Fasciola hepatica somatic antigen, its partially purified fractions and excretion-secretion products were investigated as to serological, electrophoretic and biological properties. In a Sephadex G-100 column (SG-100), Fasciola hepatica total antigen (FhTA) gave 5 fractions, and SDS-PAGE analysis showed they were glycoproteins ranging from 14 to 94 kDa molecular weight (MW). When these fractions were analyzed by enzyme linked immunotransfer blot (EITB) and immunodiffusion in gel (ID) with serum from immunized rats with FhTA, the presence of different antigenic components was revealed. In the SDS-PAGE of excretor-secretor antigen (ESA), it was possible to observe peptides from 12 to 22 kDa, which were also present in FhTA. When the FhTA, its fractions and the ESA were analyzed by EITB with the immune rat serum (IRS), it was observed that only some fractions of the SG-100 shared antigens with the FhTA and ESA. Moreover, DTH and ITH responses were studied in FhTA immunized rats challenged with these different antigen components, revealing that the protein/carbohydrate ratio is important for inducing DTH response. The ESA was the most active component in the DTH and ITH response.

Herpes simplex virus type 2 infection in pregnancy: asymptomatic viral excretion at delivery and seroepidemiologic survey of two socioeconomically distinct populations in São Paulo, Brazil

The objective of the present study was to estimate the prevalence of herpes simplex virus type 2 (HSV 2) antibodies in child bearing women of 2 Brazilian populations with different socioeconomic status and to determine the risk of neonatal HSV exposure by means of maternal cultures at the onset of labor. The study was conducted at 2 hospitals: A, serving very low income patients and B, serving middle socioeconomic class. 173 participants from group A and 127 from B answered a questionnaire which showed that the patients had similar ages (27.7 and 26.8 years, respectively) but differed with regard to socioeconomic status, age at first intercourse (18.6 vs 20.6 years), number of sex partners (1.5 vs 1.2) and previous sexually transmitted diseases (15% vs. 1.5%). History of genital herpes was given by 11% of group A participants and by a similar number, 7%, of patients from group B. In addition, 200 serum samples from population A and 455 from B were tested by ELISA for and HSV antibodies and 92% and 86%, respectively, were found to be positive. Sixty seropositive samples from group A and 90 from B were further analyzed by Western blot, which showed the presence of type 2 specific antibodies in 46% and 36%, respectively, suggesting an overall HSV 2 prevalence of 42% in group A and 31% in B. Cervical specimens were obtained for culture from 299 asymptomatic patients of population A and 313 of B. HSV was isolated from one specimen in each group, indicating a 0.3% incidence of asymptomatic viral excretion in both populations. In conclusion, the prevalence of type 2 antibodies in childbearing women was very high, but it did not differ with the socioeconomic status. The risk of HSV perinatal transmission was also similar in the 2 study populations and it was comparable with the data from developed countries. Our findings do not indicate the need of special screening programs for asymptomatic HSV excretion in Brazilian pregnant women.

The diagnostic importance of species specific and cross-reactive components of Taenia solium, Echinococcus granulosus, and Hymenolepis nana

Sera from patients infected with Taenia solium, Hymenolepis nana and Echinococcus granulosus were tested against homologous and heterologous parasite antigens using an ELISA assay, and a high degree of cross-reactivity was verified. To identify polypeptides responsible for this cross reactivity, the Enzyme Linked Immunoelectro Transfer Blot (EITB) was used. Sera from infected patients with T.solium, H.nana, and E.granulosus were assessed against crude, ammonium sulphate precipitated (TSASP), and lentil-lectin purified antigens of T.solium and crude antigens of.H.nana and E.granulosus. Several bands, recognized by sera from patients with T.solium, H.nana, and E.granulosus infections, were common to either two or all three cestodes. Unique reactive bands in H.nana were noted at 49 and 66 K-Da and in E.granulosus at 17-21 K-Da and at 27-32 K-Da. In the crude cysticercosis extract, a specific non glycoprotein band was present at 61-67 K-Da in addiction to specific glycoprotein bands of 50, 42, 24, 21, 18, 14, and 13 K-Da. None of the sera from patients with H.nana or E.granulosus infection cross reacted with these seven glycoprotein bands considered specific for T.solium infection.

Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (Viannia) braziliensis

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

DETECTION OF HTLV-I ANTIBODIES AND DNA IN BLOOD SAMPLE OF A PATIENT WITH MYELOPATHY IN NIGERIA

We describe a case of human T-lymphotropic virus type I associated myelopathy in a 50-year old woman in Nigeria. The patient presented with progressive loss of tone to the two lower limbs and later inability to walk. The HTLV-I antibody presence in the plasma collected from the patient was repeatedly detected by enzyme immunoassays (Abbott HTLV-I EIA and Coulter SELECT-HTLV I/II) and confirmed by Western blot technique. In addition, HTLV-I DNA was amplified from the genomic DNA isolated from the peripheral blood mononuclear cells of the patient by the polymerase chain reaction technique. This finding is significant being the first report of association of HTLV-I with myelopathy in Nigeria.

RESEARCH OF ANTIGEN AND ANTIBODIES FROM RETROVIRUSES, CMV AND HBV AMONG PRISONERS OF THE PENITENTIARY COMPLEX OF THE REGION OF CAMPINAS, SP, BRAZIL

Some viruses of the families Retroviridae, such as Human T Lymphotropic Virus (HTLV); Herpesviridae as the Cytomegalovirus (CMV) and Hepadnaviridae such as the Hepatitis B Virus (HBV) are liable to be co-transmitted with the Human Immunodeficiency Virus (HIV). Since prisoners are exposed to several and important risk factors involved in the transmission of HIV and the above mentioned viruses, male inmates from the penitentiary complex of Campinas, SP, Brazil, including HIV + and HIV - ones, were examined for the presence of HTLV-I and/or II antibodies; IgG and IgM anti-CMV antibodies, and the research of the superficial hepatitis B antigen (HBsAg). The presence of anti-HTLV-I and/or II was determined by the Western Blot (WB) technique, whereas IgG and IgM anti-CMV and the search of HBsAg were carried out by the Microparticle Enzyme Immunoassay (MEIA-Abbott Lab).With regard to anti-HTLV-I and/or II, 58.3% (14/24-Number of positive reactions/number of sera examined) were reactive among the anti-HIV positive sera. Conversely, only 12.5% (3/24) among the HIV- negative sera showed positive reactions to HTLV-I and/or II antibodies. When looking for IgG anti-CMV percentages of 97.7% (43/44) and 95% (38/40) were obtained for anti-HIV positive and negative sera, respectively. As to IgM anti-CMV antibodies 11.36% (5/44) and 2.5% (1/40) of reactive sera were found for anti-HIV positive and negative, respectively. The HBsAg was found in 12.8% (5/39) of the sera which were anti-HIV positive.

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.

A survey of congenital Chagas’ disease, carried out at three Health Institutions in São Paulo City, Brazil

The congenital transmission of Chagas’ disease was evaluated in 57 pregnant women with Chagas’ disease and their 58 offspring. The patients were selected from three Health Institutions in São Paulo City. The maternal clinical forms of Chagas’ disease were: indeterminate (47.4%), cardiac (43.8%) and digestive (8.8%); 55 were born in endemic areas and two in São Paulo City. The transmission of Chagas’ disease at fetal level was confirmed in three (5.17%) of the 58 cases studied and one probably case of congenital Chagas’ disease. Two infected infants were born to chagasic women with HIV infection and were diagnosed by parasitolological assays (microhematocrit, quantitative buffy coat-QBC or artificial xenodiagnosis). In both cases the placenta revealed T. cruzi and HIV p24 antigens detected by immunohistochemistry. In one case, a 14-week old abortus, the diagnosis of congenital T. cruzi infection was confirmed by immunohistochemistry. The other probable infection, a 30-week old stillborn, the parasites were found in the placenta and umbilical cord. The Western blot method using trypomastigote excreted/secreted antigens of T. cruzi (TESA) was positive for IgG antibodies in 54/55 newborns and for IgM in 1/55 newborns. One of the two newborns with circulating parasites had no detectable IgG or IgM antibodies. The assessment of IgG antibodies in the sera of pregnant women and their newborns was performed by ELISA using two different T. cruzi antigens: an alkaline extract of epimastigotes (EAE) and trypomastigote excreted/secreted antigens (TESA). The analysis showed a linear correlation between maternal and newborn IgG antibody titers at birth.

Diagnosis of abdominal angiostrongyliasis by PCR from sera of patients

Abdominal angiostrongyliasis is a zoonotic infection caused by an intra-vascular nematode parasitic of wild rodents, Angiostrongylus costaricensis. No parasitological diagnosis is currently available and immunodiagnosis presents several drawbacks. Primers constructed based on a congeneric species, A. cantonensis, were able to amplify a 232 bp fragment from serum samples of 3 patients with histopathological diagnosis. Extraction was better performed with DNAzol and the specificity of the primers was confirmed by Southern blot. This disease has been diagnosed with frequency in south of Brazil, thus, this method appears like the important and unpublished alternative to improve diagnostic of disease.

Human T-cell lymphotropic virus types I and II infections in a cohort of patients with neurological disorders in Belém, Pará, Brazil

Serum- and/or- cerebrospinal fluid (CSF) samples obtained from 190 patients suffering from chronic, progressive neurological disease were screened for the presence of human T-cell lymphotropic viruses type I (HTLV-I) and type II (HTLV-II) antibodies over a six-year period (1996 to 2001) in Belém, Pará, Brazil. Patients were of both sexes (male subjects, 52%) with ages ranging from 2 to 79 years (mean, 35.9). Overall, 15 (7.9%) subjects - of whom 12 (80%) were female adults - reacted HTLV-I/II-seropositive when screened by enzyme-linked immunosorbent assay (ELISA). Serum samples from 14 of these patients were also analyzed using a recombinant Western blot (WB) assay that yielded HTLV-I-, HTLV-II-, and HTLV-I/II- reactivities for 10 (71.4%), 3 (21.4%) and 1 (7.2%) of them, respectively. The yearly rates of HTLV-I/II antibodies ranged from 2.6% (2001) to 21.7% (2000), with progressively increasing seropositivities from 1998 to 2000. Altogether, walking difficulty (n = 5 subjects), spasticity (n = 4) and leg weakness (n = 3) accounted for 80% of symptoms recorded among the 15 patients whose sera had antibodies to HTLV-I/II as detected by ELISA. These findings provide evidence that both HTLV-I and HTLV-II play a role in the development of chronic myelopathy in Belém, Pará, Northern Brazil.