RAPD analysis of the genetic diversity of mango (Mangifera indica) germplasm in Brazil.

We evaluated genetic variability of mango (Mangifera indica) accessions maintained in the Active Germplasm Bank of Embrapa Meio-Norte in Teresina, Piauí, Brazil, using RAPDs. Among these accessions, 35 originated from plantings in Brazil, six from the USA and one from India. Genomic DNA, extracted from leaf material using a commercial purification kit, was subjected to PCR with the primers A01, A09, G03, G10, N05, and M16. Fifty-five polymorphic loci were identified, with mean of 9.16 ± 3.31 bands per primer and 100% polymorphism. Application of unweighted pair group method using arithmetic average cluster analysis demonstrated five genotypic groups among the accessions examined. The genotypes Rosa 41, Rosa 48 and Rosa 49 were highly similar (94% similarity), whereas genotypes Sensation and Rosa 18 were the most divergent (only 7% similarity). The mango accessions were found to have considerable genetic variability, demonstrating the importance of analyzing each genotype in a collection in order to efficiently maintain the germplasm collection.

Molecular characterization of cajá, Spondias mombin (Anacardiaceae), by RAPD markers.

The arboreal species Spondias mombin L. (Anacardiaceae) is widely distributed in Brazil, where the fruits, known by the common name of cajá, are an important commercial commodity. We evaluated genetic variability among 32 cajá accessions of the Germplasm Collection of Embrapa Meio-Norte using RAPD technique. Reaction conditions for efficient RAPD amplifications were optimized in preliminary tests, and primers were selected from a set designed by the University of British Columbia on the basis of high levels of polymorphism and adequate band resolution. The 21 primers employed in the final analysis produced 145 fragments, 79% of which were polymorphic. Based on the RAPD data, a dendrogram was constructed using the unweighted pair group method with arithmetic mean clustering technique. The 32 cajá accessions were classified into three main groups with a mean genetic similarity of 68.8%. Group I comprised 26 accessions (74.1% similarity), and group II included five accessions (74.0% similarity), while group III consisted of one accession (BGC 06), which exhibited the lowest similarity coefficients. Accessions BGC 06 and BGC 31 were the most unrelated and, hence, most suitable for initial crossings in order to obtain high levels of segregation. We concluded, based on the repeatability and reproducibility tests, that the RAPD technique is reliable and efficient for revealing the genetic diversity of cajá accessions, which will be useful for genetic improvement programs.

Loss of hepatitis A virus antibodies after bone marrow transplantation.

Reimmunization guidelines have recommended the inactivated HAV vaccine for hematopoietic stem cell transplant (HSCT) recipients living in or traveling to areas where hepatitis A is endemic. As a shift from high to medium hepatitis A endemicity has been observed in several countries in Latin America, we conducted a retrospective study to evaluate the prevalence of hepatitis A pre-bone marrow transplant (BMT) and the loss of specific antibodies in consecutive stored serum samples from 77 BMT recipients followed up from 82 to 1530 days. The prevalence of HAV antibodies was 92.2% before BMT. As vaccine was not available in Brazil when the samples were taken, it was assumed that this prevalence reflects natural infection. Survival analysis showed that the probability of becoming seronegative was 4.5% (+/-2.6%), 7.9% (+/-3.4%), 10.1% (+/-4.0%), 23.4% (+/-9.6%) at 1, 2, 3 and 4 years after transplant, respectively. The loss of HAV antibodies was significantly associated with longer follow-up (P=0.0015), younger age (P=0.049) and acute graft-versus-host disease (P=0.035). As most reimmunization protocols start around day +365, in developing countries with similar HAV endemicity, BMT recipients should have serological screening before HAV vaccination and the inactivated vaccine should be advised to those seronegative.

Early measles vaccination in bone marrow transplant recipients.

Measles vaccination has been recommended after the second year following bone marrow transplant (BMT) in patients not receiving immunosuppressive drugs. During a measles outbreak, we vaccinated all patients after the first year of transplant, and conducted a prospective trial to evaluate safety, effectiveness and sustained immunity after early vaccination. Patients received attenuated virus vaccine between 9 and 18 months after BMT. A total of 51 patients were evaluated and 27 of them (52.9%) were receiving immunosuppressive drugs. Only mild adverse reactions were noted. Nine patients (17.6%) were susceptible (IgG< or =100 mIU/ml) at vaccination, and all seroconverted. In those immune at vaccination, a four-fold increase in measles IgG titers was found in one of 34 patients (2.9%) with specific IgG> or =200 mIU/ml compared to 14 of 17 (82.3%) with IgG<200 mIU/ml (P< 0.0001). Sustained immunity after 24 months was more likely to occur in patients with specific IgG levels< or =200 or > or =500 mIU/mL (83.4 and 100%, respectively) in comparison to patients with 200

Analgesic and anti-inflammatory activities of (-)-o benzyl cubebin, a (-)-cubebin derivative, obtained by partial synthesis.

The anti-inflammatory and antinociceptive effects of the benzylated cubebin derivative, obtained by reaction of (-)-cubebin with benzyl bromide, were investigated using different animal models. The (-)-o-benzyl cubebin showed a low anti-inflammatory effect (16.2%) in relation to cubebin (57%) and indomethacin (77%) in the carrageenin-induced paw edema in rats, but on the other hand it was more effective (80%) than (-)-cubebin (41%) in inhibiting acetic acid-induced writhing in mice, producing dose-response correlation with doses of 10, 20 and 40 mg/kg, respectively. Moreover, this derivative compound did not show activity in both the hot plate and the cell migration test in rats. Overall, the results showed that the benzylation of cubebin were efficient in enhancing only its analgesic activity.

Analgesic and anti-inflammatory activities evaluation of (-)-O-acetyl, (-)-O-methyl, (-)-O-dimethylethylamine cubebin and their preparation from (-)-cubebin.

The anti-inflammatory and antinociceptive effects of the acetylated (2), methylated (3) and aminated (4) derivatives of cubebin (1), obtained by its reaction with acetic anhydride, methyl iodide and dimethylethylamine chloride, respectively, were investigated, using different animal models. The compound (2) was the most effective anti-inflammatory one in the carrageenin-induced paw edema in rats and was the only one which showed dose-response correlation for this assay with r = 0.993 and Y = 64.58x + 0.22. Besides, compounds (2) and (4) were more effective than cubebin in inhibiting acetic acid-induced writhing in mice, producing dose-response correlation with doses of 10, 20 and 30 mg/kg, respectively. Regarding the hot plate and the cell migration tests in rats, none of the four tested compounds showed activity. Overall, the results showed that the acetylation and amination of cubebin were efficient in enhancing its analgesic activity, as well as its anti-inflammatory activity.

Detection of human herpesvirus 8 DNA and antibodies to latent nuclear and lytic-phase antigens in serial samples from aids patients with Kaposi's sarcoma.

BACKGROUND: human herpesvirus 8 (HHV-8) have recently implicated in the etiology of Kaposi's sarcoma (KS), but the pathophysiologic and immunologic interactions between HHV-8 and the human host are incompletely understood. OBJECTIVE: this paper intends to present partial results of a follow-up study of KS patients, designed to investigate HHV-8 viremia and antibody response. METHODS: ninety-six paired serial samples (PBMCs and sera) were obtained from 12 aids patients with KS who received HAART prior or just after entry in the study. HHV-8 DNA was detected by nested-PCR and antibodies to HHV-8 latent nuclear antigen (LANA) and lytic antigen by immunofluorescence assay (IFA). RESULTS: HHV-8 DNA was detected in 33.3% of the first PBMC samples. Among the eight PCR negative patients, four presented positive samples during the follow-up and four remained negative. Five patients had intermittent viremia. Fifteen of the 96 PBMC samples were PCR positive (15.6%). Four of 39 samples (10.2%) from patients classified as stadio II and 11 of the 53 samples (20.7%) from patients in stadio IV were PCR positive (P=0.2). Six patients (50%) had anti-LANA antibodies at the entry in the study. Among the six seronegative patients, two seroconverted 2 months later and four patients remained seronegative during the 5-8 months of follow-up. All patients had anti-lytic antibodies since the first sample. CONCLUSION: the presence of HHV-8 viremia could be related to the severity of KS and could be intermittent even under HAART. A longer follow-up is needed to confirm these results.

Duality of patterns in hepatitis A epidemiology: a study involving two socioeconomically distinct populations in Campinas, São Paulo State, Brazil.

To evaluate the prevalence of antibodies against hepatitis A in two socioeconomically distinct populations, 101 and 82 serum samples from high and low socioeconomic groups, respectively, were analysed for the presence of IgG anti-HAV using a commercial ELISA. The prevalence in low socioeconomic level subjects was 95.0%, whereas in high socioeconomic subjects was only 19.6% (p < 0.001). These data show a duality in Brazil: anti-HAV prevalence in low socioeconomic subjects is similar to that of developing countries, while in high socioeconomic subjects, a pattern typical of developed countries is found. The control of this infection in our country is primarily related to the improvement of sanitation, but especially for high socioeconomic level populations, the use of vaccination against hepatitis A is strongly advisable to avoid the occasional appearance of this disease in adults.

Detection and direct typing of herpes simplex virus in perianal ulcers of patients with AIDS by PCR.

The presence of herpes simplex virus type 1 (HSV-1) and HSV-2 in perianal ulcerations of 41 AIDS patients was assessed by virus culture and a type-specific PCR-based assay. HSV was isolated from the lesion site in 24 of 41 (58.5%) patients, and HSV DNA was detected by PCR in all 24 (100%) of these specimens. Additionally, PCR was used to detect HSV DNA in 12 of 17 (70.5%) HSV culture-negative samples. Thus, HSV genomic sequences could be demonstrated in 36 of 41 (87.8%) perianal ulcers in this series. Full agreement in HSV typing by either immunodot assay or PCR was seen in 24 samples that were positive by both virus culture and PCR. HSV-2 was demonstrated in 35 of 36 (97.2%) HSV-positive samples.

Prevalence of herpes simplex type 2 antibodies and a clinical history of herpes in three different populations in Campinas City, Brazil.

OBJECTIVES: To determine the seroprevalence of herpes simplex virus type 2 (HSV-2) antibodies and the relation between the history of clinical herpes and the presence of type-specific HSV-2 antibodies in three different populations from the city of Campinas City, Brazil. POPULATION AND METHODS: One hundred and one college students, 96 patients with sexually transmitted diseases (STD), and 102 women at delivery were interviewed and blood samples were collected. Total HSV (HSV-1 and HSV-2) antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and type-specific HSV-2 antibodies were detected by Western blot assay. RESULTS: Herpes simplex virus antibodies were detected in 66.3% of the students, 97.1% of the women at delivery, and 99.0% of the STD patients. Type-specific HSV-2 antibodies were detected in 6.9% of the students, 22.6% of the women at delivery, and in 53.1% of the STD patients. History of genital herpes was reported by none of the students, by one of the women at delivery, and by 11 of 51 (21.6%) STD patients who were HSV-2 seropositive. Four of the 45 (8.9%) seronegative STD patients reported a history of genital herpes. CONCLUSION: The prevalence of HSV-2 infection in Campinas City can be significantly affected by the characteristics of the population studied, as was shown in previous studies. The sensitivity of the history of genital herpes was low in the present series, stressing that prophylactic measures for vertical and horizontal transmission of HSV-2 should not be based only on a positive history of genital ulcers.

Enzyme-linked immunosorbent assay-IgG antibody avidity test for single sample serologic evaluation of measles vaccines.

A measles-specific enzyme-linked immunosorbent assay (ELISA)-IgG avidity test for serologic evaluation of the efficacy of measles vaccines with only one blood sample was evaluated after vaccination with three measles vaccine strains. Avidity indices were determined by the urea elution technique in samples presenting antibody titers > or = 100 mIU/ml. All 127 sera collected 2-8 weeks after primary vaccination with Biken-CAM70 measles vaccine had low avidity indices (LAI, when < or = 29%) with a time-dependent increase in avidity. In samples collected 6-10 weeks after vaccination with Edmonston-Zagreb, LAI were also observed in all 31 sera tested (mean = 15%) and in 233/242 (96.3%) filter paper samples from primary vaccination with Schwarz vaccine (mean = 14%). There was no difference in the mean avidity among the three groups of primary vaccinees, although the Schwarz group had higher antibody titers. In contrast, only 1/36 (2.8%) serum samples from children who were seropositive at the time of measles vaccination had LAI (mean = 56%), despite the fact that they were collected early (2-5 weeks after vaccination). Of 90 serum samples from children vaccinated in the past with two doses and of 42 cord blood serum samples, none had LAI. It is concluded that this test is a good tool for evaluating serologically the efficacy of a single dose schedule of measles vaccine. With only one postvaccination sample, the test can discriminate nonresponders (antibody titers below 100 mIU/ml), primary responders (antibody titers > or = 100 mIu/ml with LAI), and those previously immunized (antibody titers > or = 100 mIU/ml with high avidity indices). The seroconversion rate can be calculated after excluding the latter.

[Comparison of seroconversion rates between well-nourished and malnourished children after measles immunization at nine months of age]. / Viragem sorológica após a vacinação contra o sarampo, realizada em crianças eutróficas e desnutridas, aos nove meses de idade.

The study was based on activities developed at Embu, SP, between October 1989 and June 1990. Its purpose was to study serological turning after child vaccination against measles at the age of nine months. Two groups were compared, both within the same age limits. Group number I included eutrophic children and group number II included undernourished children. Gomes criteria was used to evaluate the childrens nutritional state. Antibodies (AB) dosage was done through hemagglutination inhibition (HI) and ELISA. These two laboratory methods were also checked regarding its sensibility. Out of 130 children studied, 80 could be evaluated. From this total, 56 (70%) belonged to group I and 24 (30%) belonged to group II. When the ELISA method was used, a significantly higher seroconversion percentage (P < 0.05 or 5%) was found among children belonging to group II. This percentage was not detected when the HI method was used.

Enzyme linked immunosorbent assay for rubella antibodies: a simple method of antigen production. A preliminary report.

A simple method of rubella antigen production by treatment with sodium desoxycholate for use in enzyme immunoassay (IMT-ELISA) is presented. When this assay was compared with a commercial test (Enzygnost-Rubella, Behring), in the study of 108 sera and 118 filter paper blood samples, 96.9% (219/226) overall agreement and correlation coefficient of 0.90 between absorbances were observed. Seven samples showed discordant results, negative by the commercial kit and positive by our test. Four of those 7 samples were available, being 3 positive by HI.

Salivary antibody detection in epidemiological surveys: a pilot study after a mass vaccination campaign against rubella in São Paulo, Brazil.

The sensitivity and specificity of salivary rubella antibody detection was investigated using samples collected from 301 children after a mass vaccination campaign in the state of São Paulo, Brazil. Saliva samples were collected by 2 different methods: directly dribbling into a container or using a commercial collecting device. Corresponding finger-prick blood samples were collected on filter paper. Rubella specific immunoglobulin G (IgG) was measured in saliva by antibody capture radioimmunoassay and in blood samples by indirect enzyme-linked immunosorbent assay. The detection of salivary rubella specific IgG showed good correlation with the detection of rubella antibody in the blood samples. For both collecting techniques the predictive value for a positive saliva test was > 99% compared with the results from the blood tests. However, the predictive value for a negative saliva test was only 58.3% for a dribbled sample, compared to 100% for saliva collected using the commercial device. Moreover, collecting saliva by dribbling from children less than 4 years old was difficult. The detection of rubella specific IgG in saliva collected using a commercial device proved to be sensitive and specific in this epidemiological study, encouraging its more widespread application as a means of surveillance after mass vaccination.

Measles serodiagnosis: standardization and evaluation of a Dot-ELISA.

A Dot-ELISA using a measles virus (MV) antigen obtained by sodium deoxycholate treatment was standardized and evaluated for IgM and IgG antibody detection in measles patients and measles-vaccinated subjects. A total of 192 serum samples were studied, comprising 47 from patients with acute and convalescent measles, 55 from 9-month old children prior to measles vaccination and 41 from children of the same age after vaccination, and 49 from patients with unrelated diseases. The diagnostic performances of the IgG Dot-ELISA and IgG immunofluorescence test (IFT) were found to be close, varying from 0.97 to 1.00 in sensitivity and the specificities were maximum (1.00). Nevertheless, the sensitivity of the IgM Dot-ELISA (0.85) was higher than that (0.63) of the IgM IFT, although both assays had comparably high (1.00) specificities. The IgM Dot-ELISA in particular proved to be more sensitive in relation to other assays studied by revealing antibodies in 80.0% (12/15) of vaccinated children on the 15th day after immunization. In contrast, the IgM IFT, failed to detect antibodies in the same group of vaccinated children. The stability of the MV antigen was longer than that of the IFT antigen, and the reproducibility of the Dot-Elisa was satisfactory.

Nectomys squamipes (Rodentia: Cricetidae) as an experimental model for Schistosomiasis mansoni.

Twenty specimens of Nectomys squamipes born in captivity, were infected with 500 cercariae by the transcutaneous route. Coprologic examinations were carried out from the 5th to 23rd week after infection. On the 7th, 8th, 12th, 16th, and 23rd weeks the animals were sacrificed and perfused. The oogram was performed in segments of the small intestine (proximal, medial and distal portions) and the large intestine. The average pre-patent period was of 42 days. The average number of eggs varied from 350 on the 6th week, to 800 on the 13th. From the 14th week on, the average number of eggs eliminated was lower than 50 per gram of feces. The recovery of worms kept steady on the 7th, 8th and 12th week (16.85%; 15.45% and 11.95%), decreasing to 7.70% on the 16th week and 8.45% on the 23rd week. The proportion of male/female worms was about the same on the first two weeks, but from the 12th week on, the proportion was: 1.4/1 on the 12th week; 2.5/1 on the 16th week and 1.8/1 on the 23rd week. These observations suggest that N. squamipes may be used as an experimental model for schistosomiasis mansoni, to which it develops resistance mechanism, useful for immunity studies.

Enzyme-linked immunosorbent assay (ELISA) for measles antibody. A comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests.

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copositivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.

[Serological evaluation of immunization against measles with 2 doses administered at 6 and 11 months of age: a prospective study]. / Avaliação sorológica de imunização contra o sarampo com duas doses administradas aos 6 e 11 meses de idade: estudo prospectivo.

A prospective study was designed to evaluate the serologic efficacy of a two dose measles vaccination schedule, at 6 months and 11 months of age. Infants were given a further attenuated measles virus vaccine (BIKEN CAM 70, Fundação Oswaldo Cruz) and serum samples were tested for measles antibodies using the indirect immunofluorescence technique (IFA) and ELISA. Seroconversion rates 6 to 12 months (mean of 8.0 +/- 1.7 months) following the second dose of measles vaccine were 88.5% (85/96) by IFA and 96.8% (93/96) by ELISA. No measles cases were reported during the study period. In regions where a significant proportion of measles cases occurs before nine months of age, vaccination with a two doses schedule, at 6 and 11 months of age, may represent an alternative for measles control.

Comparison of indirect immunofluorescence test for measles antibodies with haemagglutination inhibition and plaque neutralization tests.

Indirect Immunofluorescence (IFA), Plaque Reduction Neutralization (PRN) and Haemagglutination Inhibition (HI) tests for measles antibodies were carried out in 197 sera obtained from umbilical cord and vaccinated children. The IFA was also applied to blood samples collected with filter paper. IFA results demonstrated that the test is relatively simple to perform, with good reproducibility for different antigen lots. Good correlation was obtained between IFA, PRN and HI antibody titers. Better correlation was demonstrated with IFA and PRN than with HI and PRN tests. Sensitivity of IFA in detecting antibody was less effective than PRN, however more effective than HI using rhesus monkey red blood cells. PRN antibody titers over 100 were detected by IFA but not by HI (9.7% with negative results). IFA may be of considerable practical use and able to substitute HI in seroepidemiological surveys and to evaluate vaccine efficacy. It also can be simplified by employing filter paper collected samples.