High frequency of the CCR5delta32 variant among individuals from an admixed Brazilian population with sickle cell anemia

Homozygous sickle cell disease (SCD) has a wide spectrum of clinical manifestations. In Brazil, the main cause of death of individuals with SCD is recurrent infection. The CCR5delta32 allele, which confers relative resistance to macrophage-tropic HIV virus infection, probably has reached its frequency and world distribution due to other pathogens that target macrophage in European populations. In the present investigation a relatively higher prevalence (5.1%) of the CCR5delta32 allele was identified, by PCR amplification using specific primers, in 79 SCD patients when compared to healthy controls (1.3%) with the same ethnic background (Afro-Brazilians). Based on a hypothesis that considers SCD as a chronic inflammatory condition, and since the CCR5 chemokine receptor is involved in directing a Th1-type immune response, we suggest that a Th1/Th2 balance can influence the morbidity of SCD. If the presence of the null CCR5delta32 allele results in a reduction of the chronic inflammation state present in SCD patients, this could lead to differential survival of SCD individuals who are carriers of the CCR5delta32 allele. This differential survival could be due to the development of less severe infections and consequently reduced or less severe vaso-occlusive crises.

Hepatitis C virus infection in a Brazilian population with sickle-cell anemia

Patients with sickle-cell anemia submitted to frequent blood transfusions are at risk of contamination with hepatitis C virus (HCV). Determination of HCV RNA and genotype characterization are parameters that are relevant for the treatment of the viral infection. The objective of the present study was to determine the frequency of HCV infection and the positivity for HCV RNA and to identify the HCV genotype in patients with sickle-cell anemia with a history of blood transfusion who had been treated at the Hospital of the HEMOPE Foundation. Sera from 291 patients were tested for anti-HCV antibodies by ELISA 3.0 and RIBA 3.0 Chiron and for the presence of HCV RNA by RT-PCR. HCV genotyping was performed in 19 serum samples. Forty-one of 291 patients (14.1%) were anti-HCV positive by ELISA and RIBA. Both univariate and multivariate analysis showed a greater risk of anti-HCV positivity in those who had started a transfusion regime before 1992 and received more than 10 units of blood. Thirty-four of the anti-HCV-positive patients (34/41, 82.9%) were also HCV RNA positive. Univariate analysis, used to compare HCV RNA-negative and -positive patients, did not indicate a higher risk of HCV RNA positivity for any of the variables evaluated. The genotypes identified were 1b (63%), 1a (21%) and 3a (16%). A high prevalence of HCV infection was observed in our patients with sickle-cell anemia (14.1%) compared to the population in general (3%). In the literature, the frequency of HCV infection in sickle-cell anemia ranges from 2 to 30%. The serological screening for anti-HCV at blood banks after 1992 has contributed to a better control of the dissemination of HCV infection. Because of the predominance of genotype 1, these patients belong to a group requiring special treatment, with a probable indication of new therapeutic options against HCV.

Dietary cellulose has no effect on the regeneration of hemoglobin in growing rats with iron deficiency anemia

The objective of the present study was to determine the effect of cellulose on intestinal iron absorption in rats during recovery from iron deficiency anemia. Twenty-one-day-old male Wistar-EPM rats were fed an iron-free ration for two weeks to induce anemia. At 5 weeks of age, the rats were divided into two groups (both groups receiving 35 mg of elemental iron per kg diet): cellulose group (N = 12), receiving a diet containing 100 g of cellulose/kg and control (N = 12), receiving a diet containing no cellulose. The fresh weight of the feces collected over a 3-day period between the 15th and 18th day of dietary treatment was 10.7 ± 3.5 g in the group receiving cellulose and 1.9 ± 1.2 g in the control group (P<0.001). Total food intake was higher in the cellulose group (343.4 ± 22.0 g) than in the control (322.1 ± 13.1 g, P = 0.009) during the 3 weeks of dietary treatment. No significant difference was observed in weight gain (cellulose group = 132.8 ± 19.2, control = 128.0 ± 16.3 g), hemoglobin increment (cellulose group = 8.0 ± 0.8, control = 8.0 ± 1.0 g/dl), hemoglobin level (cellulose group = 12.3 ± 1.2, control = 12.1 ± 1.3 g/dl) or in hepatic iron levels (cellulose group = 333.6 ± 112.4, control = 398.4 ± 168.0 µg/g dry tissue). We conclude that cellulose does not adversely affect the regeneration of hemoglobin, hepatic iron level or the growth of rats during recovery from iron deficiency anemia.

ßS-Haplotypes in sickle cell anemia patients from Salvador, Bahia, Northeastern Brazil

ßS-Globin haplotypes were studied in 80 (160 ßS chromosomes) sickle cell disease patients from Salvador, Brazil, a city with a large population of African origin resulting from the slave trade from Western Africa, mainly from the Bay of Benin. Hematological and hemoglobin analyses were carried out by standard methods. The ßS-haplotypes were determined by PCR and dot-blot techniques. A total of 77 (48.1%) chromosomes were characterized as Central African Republic (CAR) haplotype, 73 (45.6%) as Benin (BEN), 1 (0.63%) as Senegal (SEN), and 9 (5.63%) as atypical (Atp). Genotype was CAR/CAR in 17 (21.3%) patients, BEN/BEN in 17 (21.3%), CAR/BEN in 37 (46.3%), BEN/SEN in 1 (1.25%), BEN/Atp in 1 (1.25%), CAR/Atp in 6 (7.5%), and Atp/Atp in 1 (1.25%). Hemoglobin concentrations and hematocrit values did not differ among genotype groups but were significantly higher in 25 patients presenting percent fetal hemoglobin (%HbF) > or = 10% (P = 0.002 and 0.003, respectively). The median HbF concentration was 7.54 ± 4.342% for the CAR/CAR genotype, 9.88 ± 3.558% for the BEN/BEN genotype, 8.146 ± 4.631% for the CAR/BEN genotype, and 4.180 ± 2.250% for the CAR/Atp genotype (P = 0.02), although 1 CAR/CAR individual presented an HbF concentration as high as 15%. In view of the ethnic and geographical origin of this population, we did not expect a Hardy-Weinberg equilibrium for CAR/CAR and BEN/BEN homozygous haplotypes and a high proportion of heterozygous CAR/BEN haplotypes since the State of Bahia historically received more slaves from Western Africa than from Central Africa.

Use of molecular epidemiology to monitor the nosocomial dissemination of methicillin-resistant Staphylococcus aureus in a University Hospital from 1991 to 2001

Methicillin-resistant Staphylococcus aureus (MRSA) has been the cause of major outbreaks and epidemics among hospitalized patients, with high mortality and morbidity rates. We studied the genomic diversity of MRSA strains isolated from patients with nosocomial infection in a University Hospital from 1991 to 2001. The study consisted of two periods: period I, from 1991 to 1993 and period II from 1995 to 2001. DNA was typed by pulsed-field gel electrophoresis and the similarity among the MRSA strains was determined by cluster analysis. During period I, 73 strains presented five distinctive DNA profiles: A, B, C, D, and E. Profile A was the most frequent DNA pattern and was identified in 55 (75.3%) strains; three closely related and four possibly related profiles were also identified. During period II, 80 (68.8%) of 117 strains showed the same endemic profile A identified during period I, 18 (13.7%) closely related profiles and 18 (13.7%) possibly related profiles and, only one strain presented an unrelated profile. Cluster analysis showed a 96% coefficient of similarity between profile A from period I and profile A from period II, which were considered to be from the same clone. The molecular monitoring of MRSA strains permitted the determination of the clonal dissemination and the maintenance of a dominant endemic strain during a 10-year period and the presence of closely and possibly related patterns for endemic profile A. However, further studies are necessary to improve the understanding of the dissemination of the endemic profile in this hospital.

Morphological and functional alterations of the intestine of rats with iron-deficiency anemia

The present study was designed to assess the intestinal absorption of D-xylose and jejunal morphometry in rats with iron-deficiency anemia. Male Wistar rats were randomly divided into a control group (diet containing 50 mg Fe/kg, N = 12) and an anemic group (diet containing <5 mg Fe/kg, N = 12). The animals were housed in individual metabolic cages and deionized water and diet were provided ad libitum for 6 weeks. Hemoglobin and hematocrit were determined at 0, 2, 4, and 6 weeks. At the end of the study the rats were submitted to a D-xylose absorption test (50 mg/100 g body weight) and sacrificed and a jejunal specimen was obtained for morphometric study. At the end of the study the hemoglobin and hematocrit of the anemic rats (8.7 ± 0.9 g/dl and 34.1 ± 2.9%, respectively) were significantly (P < 0.05) lower than those of the controls (13.9 ± 1.4 g/dl and 47.1 ± 1.5%, respectively). There was no statistical difference in D-xylose absorption between the anemic (46.5 ± 7.4%) and control (43.4 ± 9.0%) groups. The anemic animals presented statistically greater villus height (445.3 ± 36.8 µm), mucosal thickness (614.3 ± 56.3 µm) and epithelial surface (5063.0 ± 658.6 µm) than control (371.8 ± 34.3, 526.7 ± 62.3 and 4401.2 ± 704.4 µm, respectively; P < 0.05). The increase in jejunum villus height, mucosal thickness and epithelial surface in rats with iron-deficiency anemia suggests a compensatory intestinal mechanism to increase intestinal iron absorption.

Frequency of Fanconi anemia in Brazil and efficacy of screening for the FANCA 3788-3790del mutation

Fanconi anemia (FA) is an autosomal recessive genetic disease characterized by progressive bone marrow failure, susceptibility to cancer and multiple congenital anomalies. There is important clinical variability among patients and the knowledge of factors which might predict outcome would greatly help the decision making regarding the choices of treatment and the appropriate time to start it. Future studies of the possible correlation between specific mutations with specific clinical presentations will provide the answer to one of these factors. At our Center we standardized a rapid and precise screening test using a mismatch PCR assay for a specific mutation (3788-3790del in exon 38 of gene FANCA) in Brazilian FA patients. We present the results obtained after screening 80 non-consanguineous FA patients referred from all regions of Brazil with a clinical diagnosis of FA supported by cellular hypersensitivity to diepoxybutane. We were able to detect the 3788-3790del allele in 24 of the 80 (30%) FA patients studied. Thirteen of the 80 (16.25%) were homozygotes and 11 of the 80 (13.75%) were compound heterozygotes, thus confirming the high frequency of the FANCA 3788-3790del mutation in Brazilian FA patients. The identification of patients with specific mutations in the FA genes may lead to a better clinical description of this condition, also providing data for genotype-phenotype correlations, to a better understanding of the interaction of this specific mutation with other mutations in compound heterozygote patients, and ultimately to the right choices of treatment for each patient with improvement of the prognosis on future studies.

Molecular epidemiology of type 1 and 2 dengue viruses in Brazil from 1988 to 2001

Dengue is a mosquito-borne viral infection that in recent decades has become a major international public health concern. Epidemic dengue fever reemerged in Brazil in 1981. Since 1990 more than one dengue virus serotype has been circulating in this tropical country and increasing rates of dengue hemorrhagic fever and dengue shock syndrome have been detected every year. Some evidence supports the association between the introduction of a new serotype and/or genotype in a region and the appearance of dengue hemorrhagic fever. In order to study the evolutionary relationships and possible detection of the introduction of new dengue virus genotypes in Brazil in the last years, we analyzed partial nucleotide sequences of 52 Brazilian samples of both dengue type 1 and dengue type 2 isolated from 1988 to 2001 from highly endemic regions. A 240-nucleotide-long sequence from the envelope/nonstructural protein 1 gene junction was used for phylogenetic analysis. After comparing the nucleotide sequences originally obtained in this study to those previously studied by others, and analyzing the phylogenetic trees, we conclude that, after the initial introduction of the currently circulating dengue-1 and dengue-2 genotypes in Brazil, there has been no evidence of introduction of new genotypes since 1988. The increasing number of dengue hemorrhagic fever cases seen in Brazil in the last years is probably associated with secondary infections or with the introduction of new serotypes but not with the introduction of new genotypes.

Allogeneic hematopoietic stem cell transplantation from an alternative stem cell source in Fanconi anemia patients: analysis of 47 patients from a single institution

We transplanted 47 patients with Fanconi anemia using an alternative source of hematopoietic cells. The patients were assigned to the following groups: group 1, unrelated bone marrow (N = 15); group 2, unrelated cord blood (N = 17), and group 3, related non-sibling bone marrow (N = 15). Twenty-four patients (51%) had complete engraftment, which was not influenced by gender (P = 0.87), age (P = 0.45), dose of cyclophosphamide (P = 0.80), nucleated cell dose infused (P = 0.60), or use of anti-T serotherapy (P = 0.20). Favorable factors for superior engraftment were full HLA compatibility (independent of the source of cells; P = 0.007) and use of a fludarabine-based conditioning regimen (P = 0.046). Unfavorable factors were > or = 25 transfusions pre-transplant (P = 0.011) and degree of HLA disparity (P = 0.007). Intensity of mucositis (P = 0.50) and use of androgen prior to transplant had no influence on survival (P = 0.80). Acute graft-versus-host disease (GVHD) grade II-IV and chronic GVHD were diagnosed in 47 and 23% of available patients, respectively, and infections prevailed as the main cause of death, associated or not with GVHD. Eighteen patients are alive, the Kaplan-Meyer overall survival is 38% at ~8 years, and the best results were obtained with related non-sibling bone marrow patients. Three recommendations emerged from the present study: fludarabine as part of conditioning, transplant in patients with <25 transfusions and avoidance of HLA disparity. In addition, an extended family search (even when consanguinity is not present) seeking for a related non-sibling donor is highly recommended.

Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

The fetal hemoglobin (HbF) levels and ßS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2%) patients had CAR/Ben genotype; 36 (28.8%) Ben/Ben; 18 (14.4%) CAR/CAR; 2 (1.6%) CAR/Atypical; 2 (1.6%) Ben/Cam; 1 (0.8%) CAR/Cam; 1 (0.8%) CAR/Arab-Indian, and 1 (0.8%) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L-) was not detected in 77 patients (91.7%). In 2 patients (2.4%), there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L-) and 5 patients (5.9%) had both isoforms (FANCD2S+/FANCD2L+). This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84) and specificity of 100% (98/98). This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L-) or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-), to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+) require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

A retrospective comparison of cyclophosphamide plus antithymocyte globulin with cyclophosphamide plus busulfan as the conditioning regimen for severe aplastic anemia

Allogeneic hematopoietic stem cell transplantation (AHSCT) is the treatment of choice for young patients with severe aplastic anemia (SAA). The association of antithymocyte globulin (ATG) and cyclophosphamide (CY) is the most frequently used conditioning regimen for this disease. We performed this retrospective study in order to compare the outcomes of HLA-matched sibling donor AHSCT in 41 patients with SAA receiving cyclophosphamide plus ATG (ATG-CY, N = 17) or cyclophosphamide plus busulfan (BU-CY, N = 24). The substitution of BU for ATG was motivated by the high cost of ATG. There were no differences in the clinical features between the two groups, including age, gender, cytomegalovirus status, ABO match, interval between diagnosis and transplant, and number of total nucleated cells infused. No differences were observed in the time to neutrophil and platelet engraftment, or in the risk of veno-occlusive disease and hemorrhage. However, there was a higher risk of mucositis in the BU-CY group (71 vs 24%, P = 0.004). There were no differences in the incidence of neutrophil and platelet engraftment, acute and chronic graft-versus-host disease, and transplant-related mortality. There was a higher incidence of late rejection in the ATG-CY group (41 vs 4%, P = 0.009). Although the ATG-CY group had a longer follow-up (101 months) than the BU-CY group (67 months, P = 0.04), overall survival was similar between the groups (69 vs 58%, respectively, P = 0.32). We conclude that the association BU-CY is a feasible option to the conventional ATG-CY regimen in this population.

Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their βS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C→T in the HBG1 promoter region were associated with the Central African Republic βS-globin haplotype. In contrast, the -369 C→G and 309 A→G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

Epidemiology of recurrent venous thrombosis

Venous thrombosis, including deep vein thrombosis and pulmonary embolism, is a common disease that frequently recurs. Recurrence can be prevented by anticoagulants, but this comes at the risk of bleeding. Therefore, assessment of the risk of recurrence is important to balance the risks and benefits of anticoagulant treatment. This review briefly outlines what is currently known about the epidemiology of recurrent venous thrombosis, and focuses in more detail on potential new risk factors for venous recurrence. The general implications of these findings in patient management are discussed.

Evaluation of renal function in sickle cell disease patients in Brazil

The objective of this study was to investigate renal function in a cohort of 98 patients with sickle cell disease (SCD) followed up at a tertiary hospital in Brazil. Clinical and laboratory characteristics at the time of the most recent medical examination were analyzed. Renal function was evaluated by the estimation of glomerular filtration rate (GFR) by the criteria of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). We compared patients with normal GFR to patients with decreased GFR (<60 mL·min-1·(1.73 m²)-1) and hyperfiltration (>120 mL·min-1·(1.73 m²)-1). Comparison between patients according to the use of hydroxyurea and comparison of clinical and laboratory parameters according to GFR were also carried out. Average patient age was 33.8 ± 13.3 years (range 19-67 years), and 57 (58.1%) patients were females. The comparison of patients according to GFR showed that patients with decreased GFR (<60 mL·min-1·(1.73 m²)-1) were older, had lower levels of hematocrit, hemoglobin and platelets and higher levels of urea and creatinine. Independent risk factors for decreased GFR were advanced age (OR = 21.6, P < 0.0001) and anemia (OR = 39.6, P < 0.0001). Patients with glomerular hyperfiltration tended to be younger, had higher levels of hematocrit, hemoglobin and platelets and lower levels of urea and creatinine, with less frequent urinary abnormalities. Hydroxyurea, at the dosage of 500-1000 mg/day, was being administered to 28.5% of the patients, and there was no significant difference regarding renal function between the two groups. Further studies are required to establish the best therapeutic approach to renal abnormalities in SCD.