Plasmodium vivax From Duffy-Negative and Duffy-Positive Individuals Share Similar Gene Pools in East Africa.

Plasmodium vivax malaria was thought to be rare in Africa, but an increasing number of P. vivax cases reported across Africa and in Duffy-negative individuals challenges this dogma. The genetic characteristics of P. vivax in Duffy-negative infections, the transmission of P. vivax in East Africa, and the impact of environments on transmission remain largely unknown. This study examined genetic and transmission features of P. vivax from 107 Duffy-negative and 305 Duffy-positive individuals in Ethiopia and Sudan. No clear genetic differentiation was found in P. vivax between the 2 Duffy groups, indicating between-host transmission. P. vivax from Ethiopia and Sudan showed similar genetic clusters, except samples from Khartoum, possibly due to distance and road density that inhibited parasite gene flow. This study is the first to show that P. vivax can transmit to and from Duffy-negative individuals and provides critical insights into the spread of P. vivax in sub-Saharan Africa.

Duffy blood group system and ocular toxoplasmosis.

Duffy blood group phenotypes [Fy(a + b-), Fy(a-b+), Fy(a + b+), Fy(a-b-)], characterized by the expression of Fya, and Fyb antigens, are present in red blood cells. Therefore, we hypothesize that the non-hematopoietic expression of these antigens might influence cell invasion by T. gondii. 576 consecutive patients from both genders were enrolled. The presumed OT clinical diagnosis was performed. Duffy phenotyping was performed by hemagglutination in gel columns and for the correct molecular characterization Fy(a-b-) phenotype, using PCR-RFLP. Anti-T. gondii IgG antibodies were detected by ELISA. Chi-square, Fisher's exact tests were used to compare the proportions. OT was present in 22.9% (n = 132) and absent in 77.1% (n = 444) of patients. The frequencies of anti-T. gondii IgG antibodies were higher in OT (127/132, 96.2%) than those without this disease (321/444, 72.3%) (p < .0001). None of the Duffy antigens or phenotypes were associated with T. gondii infection (χ2: 2.222, GL: 3, p = .5276) as well as the risk of OT (χ2: 0.771, GL: 3, p = .8566). Duffy blood group system phenotypes and their antigens do not constitute risk factors for infection by T. gondii infection and the development of OT.

Distribution of Duffy Phenotypes among Plasmodium vivax Infections in Sudan.

Negative Duffy expression on the surface of human red blood cells was believed to be a barrier for Plasmodium vivax infection in most Africans. However, P. vivax has been demonstrated to infect Duffy-negative individuals in several Central and East African countries. In this study, we investigated the distribution of Duffy blood group phenotypes with regard to P. vivax infection and parasitemia in Sudan. Out of 992 microscopic-positive malaria samples, 190 were identified as P. vivax positive infections. Among them, 186 were P. vivax mono-infections and 4 were mixed P. vivax and Plasmodium falciparum infections. A subset of 77 samples was estimated with parasitemia by quantitative real-time PCR. Duffy codons were sequenced from the 190 P. vivax positive samples. We found that the Duffy Fy(a-b+) phenotype was the most prevalent, accounting for 67.9% of all P. vivax infections, while homozygous Duffy-negative Fy(a-b-) accounted for 17.9% of the P. vivax infections. The prevalence of infection in Fy(a-b+) and Fy(a+b-)were significantly higher than Fy(a-b-) phenotypes (p = 0.01 and p < 0.01, respectively). A significantly low proportion of P. vivax infection was observed in Duffy negative individuals Fy(a-b-). This study highlights the prevalence of P. vivax in Duffy-negatives in Sudan and indicates low parasitemia among the Duffy-negative individuals.

Comparative study of alloimmunization against red cell antigens in sickle cell disease & thalassaemia major patients on regular red cell transfusion.

BACKGROUND & OBJECTIVES: : Sickle cell disease (SCD) patients require red cell transfusion during different clinical complications of the disease. Such patients are at a high risk for developing alloantibody against red cell antigens. From India, there are limited data available on alloantibody formation in multiply transfused SCD patients. The present study was thus undertaken to fill up this lacunae by looking at the development of red cell alloantibodies in SCD and ß-thalassaemia patients on regular transfusion. METHODS: : All sickle cell disease patients undergoing red cell transfusion between 2008 and 2016, were included. During this period, a large number of ß-thalassaemia major patients also underwent regular red cell transfusion. These thalassaemia patients were also included to compare the tendency of antibody formation between SCD and ß-thalassaemia major patients. All patients before regular transfusion were regularly assessed for the development of red cell antibody. Red cell antigen, antibody screen crossmatch and antibody identification were done using the standard technique. RESULTS: : A total of 138 patients with SCD aged between 4 and 53 yr (mean 17.6 yr) consisting of 83 males and 55 females (male:female, 1.5:1) along with 333 transfusion-dependent ß-thalassaemia patients were studied. Over the last eight years, 15 patients with SCD and four patients with thalassaemia developed alloantibody (P <0.001). Antibody specificity of their alloantibodies was against Rhc, RhE, Kell, Fya and Fyb only. Sickle cell disease patients with and without alloantibody required on the average 11.8 and 8.6 units of red cell concentrate, respectively (P <0.05). INTERPRETATION & CONCLUSIONS: : About 11 per cent of the transfused sickle cells patients developed alloantibodies. The antibody specificity was restricted to Rh, Kell and Duffy blood group systems. Extended antigen matching involving Rh, Kell and Duffy antigens may prevent alloantibody in such patients.

Growing evidence of Plasmodium vivax across malaria-endemic Africa.

Effective malaria control strategies require an accurate understanding of the epidemiology of locally transmitted Plasmodium species. Compared to Plasmodium falciparum infection, Plasmodium vivax has a lower asexual parasitaemia, forms dormant liver-stages (hypnozoites), and is more transmissible. Hence, treatment and diagnostic policies aimed exclusively at P. falciparum are far less efficient against endemic P. vivax. Within sub-Saharan Africa, malaria control programmes justly focus on reducing the morbidity and mortality associated with P. falciparum. However, the recent emphasis on malaria elimination and increased accessibility of more sensitive diagnostic tools have revealed greater intricacies in malaria epidemiology across the continent. Since 2010, the number of studies identifying P. vivax endemic to Africa has expanded considerably, with 88 new scientific reports published since a review of evidence in 2015, approximately doubling the available data. There is evidence of P. vivax in all regions of Africa, apparent from infected vectors, clinical cases, serological indicators, parasite prevalence, exported infections, and P. vivax-infected Duffy-negative individuals. Where the prevalence of microscopic parasitaemia is low, a greater proportion of P. vivax infections were observed relative to P. falciparum. This evidence highlights an underlying widespread presence of P. vivax across all malaria-endemic regions of Africa, further complicating the current practical understanding of malaria epidemiology in this region. Thus, ultimate elimination of malaria in Africa will require national malaria control programmes to adopt policy and practice aimed at all human species of malaria.

Ethnic benign neutropenia: A phenomenon finds an explanation.

Ethnic benign neutropenia (ENP) is the most common form of neutropenia (NP) worldwide, if an absolute blood neutrophil count (ANC) of < 1.5 G/L is used as definition. In 2009, ENP was associated with a gene variation in the ACKR1/DARC gene, the same variation that also confers the Duffy-null trait. In 2017, a novel mechanism for ENP was introduced, questioning if ENP is a true neutropenic state, when the body's total neutrophil count (TBNC) is concerned. Here, we summarize the current knowledge of ENP, asking (1) How well does the peripheral blood ANC predict the TBNC? (2) Can we improve methods for assessing TBNC? (3) Will estimates of TBNC predict infection propensity and reduce the need for further, costly workup?

Maternal red blood cell alloantibodies identified in blood samples obtained from Iranian pregnant women: the first population study in Iran.

BACKGROUND: The objective was to determine the frequency of occurrence of alloantibodies among pregnant women in Iran. STUDY DESIGN AND METHODS: This was a prospective cross-sectional study, which was carried out in the immunohematology reference laboratory of the Iranian Blood Transfusion Organization in Tehran, Iran, in 2008 to 2015. Screening and identification of red blood cell (RBC) alloantibodies was done on the sera of 7340 pregnant females using the standard tube method and gel column agglutination technique. RESULTS: Alloantibodies were identified in the serum of 332 of the 7340 (4.5%) pregnant women. A total of 410 antibodies were detected in 332 positive maternal serum samples with no previous history of blood transfusion. Anti-D was the most common antibody accounting for 70.5% of all the antibodies formed in D- women. The incidence of specific alloimmunization other than Rh group was 14.4%. CONCLUSION: We concluded that the alloimmunization rate was high in comparison with wide pattern in previous studies. In Iran, like other developing countries, alloimmunization screening tests are performed only to detect anti-D in pregnant D- women. This high rate of alloimmunization, quite possibly, is due to the fact that the majority of blood samples came from pregnant women known to have previous obstetric problems. However, we suggest that RBC antibody screening tests should be extended to all D+ women.

Procoagulant and proinflammatory effects of red blood cells on lipopolysaccharide-stimulated monocytes.

BACKGROUND: We aimed to evaluate the mechanisms underlying the effects of red blood cells (RBCs) on the reactivity of monocytes to lipopolysaccharide (LPS) stimulation. METHODS: Measurements of tissue factor (TF) antigen and activity were performed on freshly isolated white blood cells (WBCs)/platelets resuspended in heparinized plasma, as well as cultured monocytic cells. RESULTS: In a dose-dependent manner, RBCs significantly enhanced LPS-induced TF activity and antigen levels in blood monocytes; potentiation of TF activity by both human and murine RBCs did not require the presence of neutrophils and/or platelets. We also measured the levels of monocyte chemotactic protein-1 (MCP-1), the key proinflammatory chemokine that binds to duffy antigen receptor for chemokines (DARC) on RBC surface, in plasma and RBC lysates after the incubation of RBCs with WBC/platelets; at the concentrations corresponding to normal blood counts, RBCs exerted a significant influence on the free plasma levels of MCP-1, with about two-thirds of detectable MCP-1 post-LPS stimulation being associated with RBCs. Critically, DARC-deficient murine RBCs failed to enhance LPS-induced TF activity, confirming the mechanistic significance of RBC-DARC. CONCLUSIONS: Our study reports a novel mechanism by which RBCs promote procoagulant and proinflammatory sequelae of WBC exposure to LPS, likely mediated by RBC-DARC in the microenvironment(s) that bring monocytes and RBCs in close proximity.

Measurement of macrophage marker in hyperhaemolytic transfusion reaction: a case report.

BACKGROUND: Hyperhaemolytic transfusion reaction (HHTR) has been well described in patients with sickle cell disease (SCD). It is characterised by a decrease in haemoglobin concentration to levels below those before transfusion and a fall in the absolute reticulocyte count. As red blood cells (RBC) alloantibodies are typically not detected in post-transfusion samples in acute forms of HHTR, we have previously proposed that both the transfused and autologous RBCs cells (HbSS/reticulocytes) are destroyed by activated macrophages. CASE REPORTS: We report a patient with SCD who presented with vaso-occlusive sickle cell crisis and developed a severe HHTR attributable to anti-Fy3. In addition to the usual supportive measures, the patient was treated with intravenous immunoglobulin (IVIG) and steroids. Serum ferritin levels were measured as an aspecific marker of macrophage activation. RESULTS: Steroids and IVIG were effective in managing HHTR. Ferritin levels were high at the time of haemolysis, (>10000 µg L(-1)) whereas recovery and cessation of haemolysis correlated with a decrease in ferritin levels. CONCLUSION: Serum ferritin values >10,000 µg L(-1) are considered pathognomic for conditions characterised by abnormal macrophage activation. In our case, serum ferritin levels correlate well with the disease activity and clinical response. This further supports our previous proposal that the activated macrophages play an important role in HHTR. Serum ferritin is a nonspecific marker of inflammation. A rapid specific bio-marker to measure the activity of macrophages in SCD in HHTR is desirable, and this area warrants further investigation.

[Preparing of a ssDNA target in a single-round PCR with low-melt excess primer for hybridization with microarrays].

Method of ssDNA preparing in single-round PCR for microarray application is described. The approach is exemplified on genotyping of DARC gene. It is opposed to two-round PCR that consists of separate symmethric and asymmethric stages. Implementation of reaction in single round is achieved by means of low-melt excess internal primer application. The primer do not anneal during symmethric stage but after decreasing of annealing temperature on asymmethric stage. The results indicate effective oligonucleotide microarray genotyping. The approach reduces time requirements and risk of contamination.

The Duffy Antigen/Receptor for Chemokines (DARC) and prostate-cancer risk among Jamaican men.

As an evolutionary response to prevent malaria infection, most Africans do not express the Duffy Antigen/Receptor for Chemokines (DARC) on their red blood cells. Results from experimental studies suggest that DARC expression inhibits prostate-tumor growth. We tested the hypothesis that men of African descent who lack DARC expression are at increased risk of prostate cancer. A case-control study involving 81 age-matched pairs was conducted in Jamaica. Participants were interviewed to collect data, and they donated blood for determination of DARC expression. Logistic regression was used to estimate associations with prostate cancer and aggressive disease. Little or no association was observed between erythrocyte DARC expression and prostate cancer or between DARC expression and aggressive disease. These associations changed little when adjusting for other potential confounders. Our results do not support an effect of erythrocyte DARC expression on the risk or progression of prostate cancer in men of African descent.

Caracterização do receptor DARC (Duffy Antigen/Receptor for Chemokines) e da resposta imune anti Duffy Binding Protein em indivíduos expostos ao Plasmodium vivax / Characterization of the receptor DARC (Duffy Antigen / Receptor for Chemokines) and the immune response anti Duffy Binding Protein in individuals exposed to Plasmodium vivax

O processo de invasão dos eritrócitos pelos plasmódios é complexo, sendo mediado por interações moleculares específicas do tipo ligante receptor. No caso do Plasmodium vivax, a invasão é altamente dependente do antígeno de grupo sanguíneo Duffy (DARC), presente na superfície dos eritrócitos, que interage com uma proteína do parasito, a Duffy binding protein (PvDBP). Considerando a importância do. receptor DARC como principal via de invasão do P. vivax, no presente estudo desenvolveu-se uma metodologia para genotipagem do receptor DARC. A técnica foi realizada através da PCR em tempo real, em sistema multiplex, o que otimizou o método de genotipagem em larga escala e reduziu o custo. Esta nova metodologia permitiu genotipar o receptor DARC dos indivíduos que participaram de um estudo. epidêmico de malária e de outros estudos com indivíduos de diferentes áreas endêmicas da região Amazônica brasileira. No presente trabalho, para avaliar a influência de DARC na infecção pelo P. vivax, duas abordagens metodológicas foram utilizadas: um estudo de prevalência e um de incidência do tipo prospectivo. Para o estudo que buscou associação de DARC com a prevalência de malária por P. vivax, foram estudados indivíduos, proveniente de outros estados brasileiros, que migraram para a Amazônia.

Nesta população foi observado que os indivíduos que possuíam dois alelos DARC funcionais apresentavam um maior risco de infecção ao P. vivax. Já no estudo prospectivo de base populacional, o receptor DARC foi genotipado em cerca de 800 indivíduos nativos da Amazônia, residentes em um assentamento agrícola na Amazônia. Embora nenhuma associação tenha sido encontrada entre o genótipo. DARC e infecção pelo P. vivax, os resultados sugeriram que o receptor DARC influenciou na resposta de anticorpos. Nesta área, indivíduos com apenas um alelo funcional (FY*B/FY*BES) apresentaram uma resposta maior de anticorpos anti-PvDBP. Estes achados são importantes, já que a relação entre DARC e a resposta de anticorpos anti-PvDBP tem sido pouco estudada. Além disso, foram determinados os. genótipos de DARC em indivíduos residentes em uma área de transmissão epidêmica de malária por P. vivax (surto ocorrido na região metropolitana de Belo Horizonte, MG), onde se avaliou também a resposta de anticorpos anti-PvDBP. Na área do surto, a caracterização dos genótipos de DARC dos indivíduos expostos à transmissão demonstrou que os diferentes genótipos estavam igualmente distribuídos entre aqueles que se infectaram e os não infectados. Por último, nesta área do surto epidêmico, estudou-se a cinética da resposta dos anticorpos anti-PvDBP. O resultados mostraram que anticorpos anti-PvDBP são de curta duração e específicos para a variante do parasito que causou o surto. Em conjunto, acredita-se que os resultados apresentados aqui possam contribuir para os estudos que visem o melhor entendimento da relação entre DARC, resposta imune e susceptibilidade a infecção pelo P. vivax.

Caracterização do receptor DARC (Duffy Antigen/Receptor for Chemokines) e da resposta imune anti Duffy Binding Protein em indivíduos expostos ao Plasmodium vivax

O processo de invasão dos eritrócitos pelos plasmódios é complexo, sendo mediado por interações moleculares específicas do tipo ligante receptor. No caso do Plasmodium vivax, a invasão é altamente dependente do antígeno de grupo sanguíneo Duffy (DARC), presente na superfície dos eritrócitos, que interage com uma proteína do parasito, a Duffy binding protein (PvDBP). Considerando a importância do. receptor DARC como principal via de invasão do P. vivax, no presente estudo desenvolveu-se uma metodologia para genotipagem do receptor DARC. A técnica foi realizada através da PCR em tempo real, em sistema multiplex, o que otimizou o método de genotipagem em larga escala e reduziu o custo. Esta nova metodologia permitiu genotipar o receptor DARC dos indivíduos que participaram de um estudo. epidêmico de malária e de outros estudos com indivíduos de diferentes áreas endêmicas da região Amazônica brasileira. No presente trabalho, para avaliar a influência de DARC na infecção pelo P. vivax, duas abordagens metodológicas foram utilizadas: um estudo de prevalência e um de incidência do tipo prospectivo. Para o estudo que buscou associação de DARC com a prevalência de malária por P. vivax, foram estudados indivíduos, proveniente de outros estados brasileiros, que migraram para a Amazônia.

Nesta população foi observado que os indivíduos que possuíam dois alelos DARC funcionais apresentavam um maior risco de infecção ao P. vivax. Já no estudo prospectivo de base populacional, o receptor DARC foi genotipado em cerca de 800 indivíduos nativos da Amazônia, residentes em um assentamento agrícola na Amazônia. Embora nenhuma associação tenha sido encontrada entre o genótipo. DARC e infecção pelo P. vivax, os resultados sugeriram que o receptor DARC influenciou na resposta de anticorpos. Nesta área, indivíduos com apenas um alelo funcional (FY*B/FY*BES) apresentaram uma resposta maior de anticorpos anti-PvDBP. Estes achados são importantes, já que a relação entre DARC e a resposta de anticorpos anti-PvDBP tem sido pouco estudada. Além disso, foram determinados os. genótipos de DARC em indivíduos residentes em uma área de transmissão epidêmica de malária por P. vivax (surto ocorrido na região metropolitana de Belo Horizonte, MG), onde se avaliou também a resposta de anticorpos anti-PvDBP. Na área do surto, a caracterização dos genótipos de DARC dos indivíduos expostos à transmissão demonstrou que os diferentes genótipos estavam igualmente distribuídos entre aqueles que se infectaram e os não infectados. Por último, nesta área do surto epidêmico, estudou-se a cinética da resposta dos anticorpos anti-PvDBP. O resultados mostraram que anticorpos anti-PvDBP são de curta duração e específicos para a variante do parasito que causou o surto. Em conjunto, acredita-se que os resultados apresentados aqui possam contribuir para os estudos que visem o melhor entendimento da relação entre DARC, resposta imune e susceptibilidade a infecção pelo P. vivax.

Benefits of blood group genotyping in multi-transfused patients from the south of Brazil.

We evaluated the usefulness of blood group genotyping as a supplement to hemagglutination to determine the red blood cell (RBC) antigen profile of polytransfused patients with hematological diseases and renal failure. Seventy-nine patients were selected. They all received more than three units of blood and eight (10%) had already clinical significant alloantibodies occurring alone or in combination against Rh, K, Fya, and Di antigens. DNA was prepared from blood samples and RHCE*E/e, KEL*01/KEL*02, FY*01/FY*02 and JK*01/JK*02 alleles were determined by using PCR-RFLP. RHD*/RHD*Ψ and RHCE*C/c were tested using multiplex PCR. Discrepancies for Rh, Kell, Duffy, and Kidd systems were found between the phenotype and genotype-derived phenotype in 16 of the 38 chronically transfused patients. The genotypes of these patients were confirmed by DNA array analysis (HEA Beadchip(™); Bioarray Solutions, Warren, NJ). Genotyping was very important for the determination of the true blood groups of the polytransfused patients, helped in the identification of suspected alloantibodies and in the selection of antigen-negative RBCs for transfusion.

In silico studies on DARC.

The Duffy Antigen/Receptor for Chemokine (DARC) is a seven segment transmembrane protein. It was firstly discovered as a blood group antigen and was the first specific gene locus assigned to a specific autosome in man. It became more famous as an erythrocyte receptor for malaria parasites (Plasmodium vivax and Plasmodium knowlesi), and finally for chemokines. DARC is an unorthodox chemokine receptor as (i) it binds chemokines of both CC and CXC classes and (ii) it lacks the Asp-Arg-Tyr consensus motif in its second cytoplasmic loop hence cannot couple to G proteins and activate their signaling pathways. DARC had also been associated to cancer progression, numerous inflammatory diseases, and possibly to AIDS. In this review, we will summarize important biological data on DARC. Then we shall focus on recent development of the elaboration and analyzes of structural models of DARC. We underline the difficulty to propose pertinent structural models of transmembrane protein using comparative modeling process, and other dedicated approaches as the Protein Blocks. The chosen structural models encompass most of the biochemical data known to date. Finally, we present recent development of protein-protein docking between DARC structural models and CXCL-8 structures. We propose a hierarchical search based on separated rigid and flexible docking.

Duffy antigen expression on reticulocytes does not alter following blood loss in an autologous donation model.

BACKGROUND: The Duffy blood group (Fy) antigen functions as the receptor whereby the malarial parasite Plasmodium vivax invades reticulocytes. In this study, we evaluated an autologous blood donation model to measure Fy expression during the anticipated response to blood loss. AIMS: This study aims to examine Fy expression following anticipated reticulocytosis in response to blood loss from autologous whole blood donation. METHOD: Subjects were healthy blood donors presenting for planned collection of two or three autologous units. Whole blood (450 ml +/- 10%) was collected and processed. Blood samples for Fy testing were obtained from the donations. These were assayed by flow cytometry by measuring binding of a phycoerythrin-labelled anti-Fy6 antibody and compared against reticulocyte numbers. Reticulocyte numbers were measured using thiazole orange. Results were compared from baseline (first donation) with samples at second and, if available, third, donations. Phenotyping for Fy a and b antigens was performed. RESULTS: Reticulocytes increased by a mean of 37% over baseline [0.93% (range 0.31-1.93) to 1.23% (0.32-3.51%)] following donation of two (n = 32) or three (n = 9) autologous whole blood units. Absolute reticulocyte count remained low. Mean and median Fy expression on mature red blood cells and reticulocytes did not change from baseline levels despite individual variation. No apparent relationship to serologically determined Fy a and/or b antigen status was present. CONCLUSION: Baseline expression of Fy antigen on mature red blood cells and reticulocytes is quite variable between individuals, but appears not to be greatly affected by mild to moderate reticulocytosis following blood loss in an autologous blood donation model.

Genetic control of resistance to human malaria.

The term 'innate resistance' covers mechanisms of resistance that operate early in the course of infections, preceding adaptive immune responses which exert effects after several days. The first example of genetically controlled innate resistance to human malaria was the demonstration in 1954 that sickle-cell heterozygotes have less severe Plasmodium falciparum infections than do children with normal adult hemoglobin. This observation has been repeatedly confirmed, most recently by independent studies of genome-wide associations in severe falciparum malaria, which have identified the HBB locus as the major signal of association. Other abnormal hemoglobins, glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency also confer some degree of resistance against falciparum malaria. A second early example of inherited innate resistance to malaria was the finding that nonexpression of the Duffy antigen/chemokine receptor (DARC) on erythrocytes confers resistance to P. vivax. However, this parasite can enter nonhuman primate red cells independently of DARC, and in some human populations P. vivax has been observed in persons lacking DARC. Hence DARC is not the only receptor for P. vivax, but it is likely to be a major one for human transmission. Innate resistance to malaria is rapidly reinforced by adaptive immune responses, both cell-mediated and humoral. Among the factors influencing the efficacy of adaptive immune responses to malaria is the MHC complex constitution of hosts. This differs among populations, presumably because of variations in the structure of parasite antigens recognized by the immune systems of hosts.