The screening of rare blood donors in a highly admixed population: A new approach for Holley and Diego genotyping and impact of genomic and self-reported ancestry.

OBJECTIVES: The present study aimed to develop strategies for genotyping DO*HY (Dombrock system) and DI*A/DI*B (Diego system) alleles and to evaluate the impact of genomic and self-declared ancestry on rare donor screening in admixed populations. BACKGROUND: The antigens Hy and Dib demonstrate clinical importance. The lack of antisera for the serological evaluation of these antigens makes it necessary to develop molecular methods. In addition, considering that some rare red blood cell phenotypes present differences in frequency between ethnic groups, it is important to assess the applicability of self-declared ancestry in the search for rare donors in admixed populations. METHODS: DO*HY and DI*A/DI*B genotyping based on real-time polymerase chain reaction (PCR) was standardised. A total of 457 blood donors clustered by self-defined skin colour/race categories were genotyped. Furthermore, individual genomic ancestry was used in the analyses. RESULTS: The assays developed are reproducible and provide satisfactory results even at low concentrations of DNA, which make them useful in situations where the DNA is scarce, such as dried blood spots on filter paper, or when screening for pooled samples. No significant difference was observed in the frequencies of the DI*A, DI*B and DO*HY, comparing the self-declared White (branco) donors with those who are Black (preto) and Brown (pardo). CONCLUSION: Real-time PCR, especially using pooled samples, is a promising strategy to screen rare blood donors. Although both self-reported race/colour and some blood group phenotypes are associated with ancestry, the results point to a greater complexity in the application of self-declared race/colour in the screening of rare donors in admixed populations.

Spinal cord hypometabolism associated with infection by human T-cell lymphotropic virus type 1(HTLV-1).

BACKGROUND: HTLV-1 infection is endemic in Brazil. About 1 to 2% of the Brazilian population is estimated to be infected, but most infected HTLV-1 individuals do not know about their own infection, which favors the continuity of sexual and vertical virus transmission. In addition, HTLV-1 associated central nervous system diseases and their pathophysiologic mechanisms are not fully understood. This study aimed to evaluate the correlation of spinal cord metabolism, viral and inflammatory profiles with features of neurological presentation in HTLV-1 infected individuals. METHODOLOGY: This is a cross-sectional study of a cohort including 48 HTLV-1 infected individuals clinically classified as asymptomatic-AG (N = 21), symptomatic-SG (N = 11) and HAM/TSP-HG (N = 16) and a nested case-control study with HTLV-1 infected individuals-HIG (N = 48) and HTLV-1 non infected controls-CG (N = 30) that had their spinal cord analysed by Positron Emission Tomography with 18F-Fluordeoxyglucose (18F-FDG PET/CT). HTLV-1 infected individuals had 18F-FDG PET/CT results analyzed with clinical and demographic data, proviral load, cytokines and chemokines in the blood and cerebrospinal fluid (CSF). PRINCIPAL FINDINGS: 18F-FDG PET/CT showed hypometabolism in the thoracic spinal cord in HTLV-1 infected individuals. The method had an accuracy of 94.4% to identify HAM/TSP. A greater involvement of the thoracic spinal cord was observed, although hypometabolism was also observed in the cervical spinal cord segment in HTLV-1 infected individuals. Individuals with HAM/TSP showed a pro-inflammatory profile in comparison to asymptomatic and symptomatic groups, with a higher level of Interferon-inducible T-cell alpha chemoattractant (ITAC/CXCL11), IL-6, IL-12p70 in the plasma; and ITAC, IL-4, IL-5, IL-8 (CXCL8) and TNF-alpha in the CSF. Using regression, thoracic spinal cord SUV (standardized uptake value) and CSF ITAC level were identified as the HAM/TSP predictors in the multivariate model. CONCLUSIONS: 18F-FDG PET/CT imaging showed spinal cord hypometabolism in most HTLV-1 infected individuals, even in the asymptomatic HTLV-1 group. Thoracic spinal cord hypometabolism and CSF-ITAC levels were identified predictors of HAM/TSP. SIGNIFICANCE: Our findings suggested that in most HTLV-1 infected individuals there was compromise of central nervous system (CNS) structures despite of the lack of clinical symptoms. To explain the found hypometabolism, the role of microcirculatory and metabolic factors in the pathogenesis of neurological diseases associated with HTLV-1 infection must be further investigated. It is paramount to evaluate the central nervous function and to compare the performance among HTLV-1 infected individuals considered asymptomatic to the uninfected controls.

Plasma and cerebrospinal fluid levels of cytokines as disease markers of neurologic manifestation in long-term HTLV-1 infected individuals.

AIM: The aim of this study is to evaluate the presence of a particular immunological profile in individuals long-term infected with HTLV-1, followed presenting different clinical courses. MATERIALS & METHODS: Forty-eight individuals were evaluated for 19 cytokines analyzed in cerebrospinal fluid and plasma of patients with HTLV-1 presenting with and without neurological symptoms. RESULTS: Proinflammatory cytokines and the chemokine ligand 11 (ITAC/CXCL11) were increased in individuals with HTLV-1 coursing with neurological symptoms. CONCLUSION: Different cytokines' expression profile in the presence of neurological symptoms may help to understand and characterize the progression for severe clinical presentations.

Genetic determinants and stroke in children with sickle cell disease / Determinantes genéticos e Acidente Vascular Encefálico em crianças com doença falciforme

Abstract Objective: To verify genetic determinants associated with stroke in children with sickle cell disease (SCD). Methods: Prospective cohort with 110 children submitted to neonatal screening by the Neonatal Screening Program, between 1998 and 2007, with SCD diagnosis, followed at a regional reference public service for hemoglobinopathies. The analyzed variables were type of hemoglobinopathy, gender, coexistence with alpha thalassemia (α-thal), haplotypes of the beta globin chain cluster, and stroke. The final analysis was conducted with 66 children with sickle cell anemia (SCA), using the chi-squared test in the program SPSS® version 14.0. Results: Among children with SCD, 60% had SCA. The prevalence of coexistence with α-thal was 30.3% and the Bantu haplotype (CAR) was identified in 89.2%. The incidence of stroke was significantly higher in those with SCA (27.3% vs. 2.3%; p = 0.001) and males (24.1% vs. 9.6%; p = 0.044). The presence of α-thal (p = 0.196), the CAR haplotype (p = 0.543), and socioeconomic factors were not statistically significant in association with the occurrence of stroke. Conclusion: There is a high incidence of stroke in male children and in children with SCA. Coexistence with α-thal and haplotypes of the beta globin chain cluster did not show any significant association with stroke. The heterogeneity between previously evaluated populations, the non-reproducibility between studies, and the need to identify factors associated with stroke in patients with SCA indicate the necessity of conducting further research to demonstrate the relevance of genetic factors in stroke related to SCD.

Resumo Objetivo: Verificar fatores genéticos associados ao acidente vascular encefálico (AVE) em crianças com doença falciforme (DF). Métodos: Coorte prospectiva de 110 crianças submetidas à triagem neonatal pelo Programa de Triagem Neonatal, entre 1998-2007, com o diagnóstico de DF, atendidas em serviço público regional de referência em hemoglobinopatias. As variáveis analisadas foram: tipo de hemoglobinopatia, sexo, coexistência da alfa-Talassemia (α-Tal), haplótipos do cluster da cadeia beta globina e AVE. A análise estatística final foi feita com 66 crianças com anemia falciforme, por meio do teste do qui-quadrado no programa SPSS® 14.0. Resultados: Entre as crianças com DF, 60% eram portadoras de anemia falciforme. A prevalência da coexistência com a α-Tal foi de 30,3% e o haplótipo Bantu (CAR) foi identificado em 89,2%. A incidência de AVE foi significativamente maior nas crianças com AF (27,3% versus 2,3%; p = 0,001) e no sexo masculino (24,1% versus 9,6%; p = 0,044. A presença da α-Tal (p = 0,196), do haplótipo CAR (p = 0,543) e de fatores socioeconômicos não foi significantemente associada à ocorrência de AVE. Conclusão: O AVE apresenta alta incidência em crianças com AF e em crianças do sexo masculino. Coexistência de α-Tal ou de haplótipos do cluster da betaglobina não apresentaram associação significante com AVE. A heterogeneticidade entre as populações previamente avaliadas e a não reprodutibilidade entre estudos indicam a necessidade de novas pesquisas para verificar o papel desses fatores genéticos no AVE em crianças com DF.

Genetic determinants and stroke in children with sickle cell disease.

OBJECTIVE: To verify genetic determinants associated with stroke in children with sickle cell disease (SCD). METHODS: Prospective cohort with 110 children submitted to neonatal screening by the Neonatal Screening Program, between 1998 and 2007, with SCD diagnosis, followed at a regional reference public service for hemoglobinopathies. The analyzed variables were type of hemoglobinopathy, gender, coexistence with alpha thalassemia (α-thal), haplotypes of the beta globin chain cluster, and stroke. The final analysis was conducted with 66 children with sickle cell anemia (SCA), using the chi-squared test in the program SPSS® version 14.0. RESULTS: Among children with SCD, 60% had SCA. The prevalence of coexistence with α-thal was 30.3% and the Bantu haplotype (CAR) was identified in 89.2%. The incidence of stroke was significantly higher in those with SCA (27.3% vs. 2.3%; p=0.001) and males (24.1% vs. 9.6%; p=0.044). The presence of α-thal (p=0.196), the CAR haplotype (p=0.543), and socioeconomic factors were not statistically significant in association with the occurrence of stroke. CONCLUSION: There is a high incidence of stroke in male children and in children with SCA. Coexistence with α-thal and haplotypes of the beta globin chain cluster did not show any significant association with stroke. The heterogeneity between previously evaluated populations, the non-reproducibility between studies, and the need to identify factors associated with stroke in patients with SCA indicate the necessity of conducting further research to demonstrate the relevance of genetic factors in stroke related to SCD.

Blocking vertical transmission of human T cell lymphotropic virus type 1 and 2 through breastfeeding interruption.

BACKGROUND: Human T cell lymphotropic virus type 1 and 2 (HTLV-1/2) causes serious diseases and is endemic in many parts of the world. It is transmitted from mother to child in 15-25% of the cases, primarily through breastfeeding. Proviral load and duration of breastfeeding are thought to play a role in transmission. This study aimed to detect HTLV-seropositive mothers through testing of neonates, to evaluate maternal HTLV proviral load and to measure the rates of transmission blocking when interruption of breastfeeding was implemented. METHODS: Neonates were screened for HTLV-1/2 IgG by enzyme immunoassay using the neonatal screening program of Minas Gerais State, Brazil. Breastfeeding interruption was recommended to those whose mothers were confirmed HTLV-positive. Children were tested by polymerase chain reaction at birth and at 12 months of age. RESULTS: Of 55,293 neonates tested, 42 (0.076%) were positive for HTLV-1 or HTLV-2 IgG. All 42 were polymerase chain reaction-negative at birth and 1 of 37 (2.7%) became antibody-positive after 12 months. His mother had delivered him vaginally and was informed of the positive HTLV-1 polymerase chain reaction after 7 days of breastfeeding. The mother's proviral load was 271 copies/10,000 cells, whereas the average is 109.2 copies/10,000 cells (95% confidence interval: 70.56-147.83). CONCLUSIONS: Maternal HTLV-1 proviral load and the route of delivery may have played a role in the transmission observed. Avoidance of breastfeeding was an effective measure to reduce HTLV transmission. In endemic countries, routine prenatal or neonatal screening combined with formula feeding for mothers confirmed HTLV-positive may be an important strategy to prevent future development of illnesses related to HTLV.

Evaluation of the effects of Quercetin and Kaempherol on the surface of MT-2 cells visualized by atomic force microscopy.

This study investigated the anti-viral effects of the polyphenolic compounds Quercetin and Kaempherol on the release of HTLV-1 from the surface of MT-2 cells. Atomic force microscopy (AFM) was used to scan the surface of the MT-2 cells. MT-2 cells were fixed with 100% methanol on round glass lamina or cleaved mica and dried under UV light and laminar flow. The images were captured on a Multimode equipment monitored by a NanoScope IIId controller from Veeco Instruments Inc operated in tapping mode and equipped with phase-imaging hardware. The images demonstrated viral budding structures 131 ± 57 nm in size, indicating profuse viral budding. Interestingly, cell-free viruses and budding structures visualized on the surface of cells were less common when MT-2 was incubated with Quercetin, and no particles were seen on the surface of cells incubated with Kaempherol. In summary, these data indicate that HTLV-1 is budding constantly from the MT-2 cell surface and that polyphenolic compounds were able to reduce this viral release. Biological samples were analyzed with crude cell preparations just after cultivation in the presence of Quercetin and Kaempherol, showing that the AFM technique is a rapid and powerful tool for analysis of antiviral activity of new biological compounds.

Uso da genotipagem de grupos sanguíneos na elucidação de casos inconclusivos na fenotipagem eritrocitária de pacientes atendidos na Fundação Hemominas / Blood group genotyping to solve inconclusive phenotyping cases of patients from the Hemominas Foundation

A determinação do perfil de antígenos eritrocitários em doadores de sangue e pacientes que recebem transfusão sanguínea é importante na prevenção de aloimunização. Pacientes recentemente transfundidos ou com anemia hemolítica autoimune nem sempre conseguem ser fenotipados, e para estes casos a genotipagem vem se apresentando como uma ferramenta auxiliar na tipagem sanguínea. Neste estudo foram padronizadas técnicas de PCR alelo específicas ou de PCR-RFLP para a genotipagem dos alelos de grupos sanguíneos Rh (RHD, RHCE*C/c, RHCE*E/e), Kell (KEL*1/KEL*2), Kidd (JK*A/JK*B) e Duffy (FY*A/FY*B e FY*B(-33T>C)), importantes na medicina transfusional. Elas foram empregadas com sucesso para a tipagem de 36 pacientes que não puderam ser fenotipados ou que apresentaram resultados inconclusivos na fenotipagem eritrocitária. Vinte destes pacientes eram aloimunizados por diferentes antígenos, sendo o anticorpo anti-E o mais frequente (55 por cento). O uso da genotipagem também mostrou-se útil na identificação de anticorpos irregulares. Por sua precisão, facilidade de execução e viabilidade de custo, as técnicas para tipagem de DNA para estes sistemas sanguíneos foram implantadas em nosso Serviço a partir de 2007 e vêm sendo usadas na prática transfusional, contribuindo para aumento da segurança dos pacientes cronicamente transfundidos ou com anemia hemolítica autoimune, como, por exemplo, pacientes com anemia falciforme. Além disso, ela vem permitindo o melhor uso de unidades de sangue com fenótipos menos frequentes na nossa população de doadores de sangue.

The determination of the blood group antigen profile of blood donors and transfusion patients is important to avoid alloimmunization. The knowledge of blood group polymorphisms acquired over the last few years has permitted the development of molecular methods that are able to predict blood group phenotypes. For patients who have recently been transfused or those who present with autoimmune hemolytic anemia, genotyping is an important tool in blood typing. We used molecular biology (allele-specific PCR and PCR-RFLP) to genotype Rh (RHD, RHCE*C/c, RHCE*E/e), Kell (KEL*1/KEL*2), Kidd (JK*A/JK*B) and Duffy (FY*A/FY*B and FYB(-33T>C)) alleles and solved the inconclusive blood types of 36 patients. Twenty patients had developed irregular antibodies of different red blood cell antigens, most frequently anti-E (55 percent). The definition of irregular antibodies was feasible by genotyping. Due to their accuracy, simplicity and economic viability, these tests have been used in the clinical practice in our Institution since 2007, contributing to the management of chronically transfused patients. Additionally, these tests allow a better use of less common blood units related to the ethnicity of the blood donor population.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) inflammatory network.

HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) is a systemic immune-mediated inflammatory disease and tissues other than nervous can be damaged, mainly ocular, rheumatic and dermatologic. Over 90% of HTLV-1-infected individuals remain lifelong asymptomatic and this retrovirus persists indefinitely in their CD4+ T-lymphocytes. The infection is maintained due to the proliferation of lymphocytes that harbor a provirus and express HTLV-1 proteins, particularly Tax, promoting an active and selective expansion of infected T cells. High proviral load is related to disease progression, which is correlated to disequilibrium between host and virus. Cytotoxic T lymphocytes are abundant and chronically activated in asymptomatic carriers and in HAM/TSP patients. The asymptomatic carriers were shown to have a high frequency of pro-inflammatory monocytes and anti-inflammatory IL-10+CD4+ and IL-10+CD8+ T-cells, as an immunoregulatory mechanism to counterbalance the monocyte-derived TNF-alpha. A putative immunomodulatory event would be the key to control their overall immunological status. In HAM/TSP, a pro-inflammatory microenvironment is the hallmark of the immunological profile. Enhanced frequency of activated CD8+ T-cells (HLA-DR+) in combination with high CD18 surface expression has been seen. In blood and cerebrospinal fluid, increased levels of Type-1 cytokines, as interferon-(IFN)-gamma, Tumor Necrosis Factor (TNF)-alpha, Interleukin (IL)-2, and pro-inflammatory IL-6, can be found. Concerning the progression, HLA polymorphisms may influence HAM/TSP and the allele HLA-A*2 has been associated with protection. The authors showed that HAM/TSP is strongly associated with a decreased percentage of B-cells, with enhanced T/B-cell ratio and activated CD8+ T-cells. These immunological parameters have been proposed as a prognostic biomarker for HAM/TSP.

Increased prevalence of human T cell lymphotropic virus type 1 in patients attending a Brazilian dermatology clinic.

Brazil may have the highest absolute number of individuals infected by human T cell lymphotropic virus type 1 (HTLV-1). It has been suggested that the prevalence of HTLV-1 is increased in patients with skin diseases. This study shows a higher prevalence of this infection in 1,229 patients attending a Brazilian dermatology clinic (0.7%) when compared to blood donors (0.22%). Of note, one additional patient tested positive for HTLV-2. The main skin diseases described in HTLV-1 seropositives were vitiligo (2 cases), dermatophytosis (2 cases), and leprosy (2 cases). A 23-year-old woman received a diagnosis of infectious dermatitis.

Infecçäo e doença pelos vírus linfotrópicos humanos de células T (HTLV-I/II) no Brasil / Infection and disease caused by the human T cell lymphotropic viruses type I and II in Brazil

A infecçäo pelos vírus HTLV-I/II encontra-se presente em todas as regiöes brasileiras, mas as prevalências variam de um estado para outro, sendo mais elevadas na Bahia, Pernambuco e Pará. As estimativas indicam que o Brasil possui o maior número absoluto de indivíduos infectados no mundo. Testes de triagem de doadores e estudos conduzidos em grupos especiais (populaçöes indígenas, usuários de drogas intravenosas e gestantes) constituem as principais fontes de informaçäo sobre essas viroses em nosso país. O HTLV-I causa a leucemia/linfoma de células T do adulto (LLTA), a paraparesia espástica tropical/mielopatia associada ao HTLV (TSP/HAM), uveíte associada ao HTLV (HAU) e anormalidades dermatológicas e imunológicas. O HTLV-II näo se mostrou associado a nenhuma doença até o momento. O diagnóstico é feito com testes de triagem (ELISA, aglutinaçäo) e confirmatórios (Western Blot, PCR). Estes vírus säo transmitidos pelo sangue e agulhas contaminadas, através de relaçöes sexuais e de mäe para filho, especialmente através do aleitamento materno. Medidas de prevençäo devem focalizar a orientaçäo de doadores soropositivos, mäes infectadas e usuários de drogas intravenosas

[Infection and disease caused by the human T cell lymphotropic viruses type I and II in Brazil]. / Infecção e doença pelos vírus linfotrópicos humanos de células T (HTLV-I/II) no Brasil.

HTLV-I/II infection is present in all regions of Brazil, but its prevalence varies according to the geographical area, being higher in Bahia, Pernambuco and Pará. It has been estimated that Brazil has the highest absolute number of infected individuals in the world. Blood donors screening and research conducted with special groups (indigenous population of Brazil, IV drug users and pregnant women) are the major sources of information about these viruses in our Country. HTLV-I causes adult T cell leukemia/lymphoma (ATLL), HTLV associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV associated uveitis (HAU), dermatological and immunological abnormalities. HTLV-II is not consistently associated with any disease. Diagnosis is established using screening (enzymatic assays, agglutination) and confirmatory (Western blot, PCR) tests. The viruses are transmitted by blood and contaminated needles, by sexual relations and from mother to child, especially by breast feeding. Prevention efforts should focus on education of positive blood donors, infected mothers and IV drug users.

HIV-1 subtyping by heteroduplex mobility assay in patients with bleeding disorders

O vírus da imunodeficiência humana do tipo 1 estabelece uma infecçäo presistente que na maioria dos casos evolui para a Síndrome de Imunodeficiência humana do tipo 1 tem sido geneticamente classificado em maior ou em outros grupos. Ogrupo maior é subdivido em nove subtipos baseados em evidências seqüênciais. A infecçäo pelo vírus da imunodeficiência humana do tipo 1 foi transmitida aos hemofílicos principalmente pelos concentrados de coagulaçäo no final da década de 70 e meados da de 80 e a síndrome da imunodeficiência adquirida tornou-se a principal causa de morbidade e morte entre estes pacientes. O Objetivo deste estudo foi o de determinar os subtipos do vírus da imunodeficiência humana em oito pacientes soropositivos com moléstias hemorrágicas de Belo Horizonte, Brasil, utilizando o ensaio de mobilidade heteroduplex, um método näo seqüêncial, que tem a propriedade de separar os portadores do vírus. Realizamos a reaçäo da cadeia de polimerase seguida pelo ensaio de mobilidade heteroduplex e obtivemos em todos os oito pacientes a confirmaçäo que os mesmos pertenciam ao subtipo B do vírus da imunodeficiência humana que é a mais prevalente nos Estados Unidos, Europa e Brasil. Os pacientes hemofílicos provavelmente foram infectados de concentrados provenientes da Europa, Estados Unidos, Säo Paulo e Rio de Janeiro (Brasil), utilizados em período anterior ao do conhecimento do vírus da imunodeficiência humana adquirida, e do uso de plasmas e concentrados näo testados, ou inativados, para o mesmo. O ensaio da mobilidade de heteroduplex e amplificaçäo do ácido desoxiribonucleico pela reaçäo de cadeia da polimerase provaram ser um modo rápido de subtipar o vírus da imunodeficiência humana adquirida em indivíduos infectados por transfusäo. O teste pode ser útil como rastreamento e detecçäo da origem e procedência do vírus da imunodeficiência adquirida.