Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification

The recent outbreak of Zika virus (ZIKV) disease caused an enormous number of infections in Central and South America, and the unusual increase in the number of infants born with microcephaly associated with ZIKV infection aroused global concern. Here, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of ZIKV. The assay specifically detected ZIKV strains of both Asian and African genotypes without cross-reactivity with other arboviruses, including Dengue and Chikungunya viruses. The assay detected viral RNA at 14.5 TCID50/mL in virus-spiked serum or urine samples within 15?min, although it was slightly less sensitive than reference real time RT-PCR assay. We then evaluated the utility of this assay as a molecular diagnostic test using 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraiba and Pernambuco, Brazil in 2016. The results of this assay were consistent with those of the reference RT-PCR test. This portable RT-LAMP assay was highly specific for ZIKV, and enable rapid diagnosis of the virus infection. Our results provide new insights into ZIKV molecular diagnostics and may improve preparedness for future outbreaks

Pathogen reduction/inactivation of products for the treatment of bleeding disorders:what are the processes and what should we say to patients?

Patients with blood disorders (including leukaemia, platelet function disorders and coagulation factor deficiencies) or acute bleeding receive blood-derived products, such as red blood cells, platelet concentrates and plasma-derived products. Although the risk of pathogen contamination of blood products has fallen considerably over the past three decades, contamination is still a topic of concern. In order to counsel patients and obtain informed consent before transfusion, physicians are required to keep up to date with current knowledge on residual risk of pathogen transmission and methods of pathogen removal/inactivation. Here, we describe pathogens relevant to transfusion of blood products and discuss contemporary pathogen removal/inactivation procedures, as well as the potential risks associated with these products: the risk of contamination by infectious agents varies according to blood product/region, and there is a fine line between adequate inactivation and functional impairment of the product. The cost implications of implementing pathogen inactivation technology are also considered

Measuring red blood cell aggregation forces using double optical tweezers

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 x 10(-3) (0.1-2.5) poise. cm. The samples analyzed with anti-D showed 2 x 10(-3) (1.0-4.0) poise. cm (p<0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction.733262264Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Bartonellosis as Cause of Death After Red Blood Cell Unit Transfusion

The authors present the case of a young man with aplastic anemia who went into shock and died after several red blood cell unit transfusions. Immunohematological studies did not show any abnormality and blood cultures from patients and blood bags were negative. The ultrastructural findings, allied with current scientific knowledge, permitted the diagnosis of Bartonella sp. infection. In face of this diagnosis, two possibilities should be considered: the first one is that the patient was already infected by the bacteria before the last RBC unit transfusion. The pathogen could be involved in aplastic anemia etiology and in the failure to recover hemoglobin levels, in spite of the transfusions. The second possibility is that the RBC unit was contaminated with a Bartonella sp., which would have led to a state of shock, causing the death of the patient.33415115

Molecular heterogeneity of the A(3) subgroup

The molecular characterization of the subgroup A(3) remains unclear. Four unrelated A(3) blood donors were studied. Family studies were possible in three of them. The A(3) subgroup was defined by immunohaematological evaluation with four different commercially available serums, Exons VI and VII of the ABO gene, responsible for 91% of the catalytic active part of the glycosyltransferase, were amplified and subjected to direct sequencing, The results in all samples showed heterozygosity for the G261 deletion, In the A(3) allele, the following associations were found: C467T mutation and 1060C deletion in one A(3) blood donor and in another G829A and 1060C. In one case, only the 1060C deletion was demonstrated in the A(3) allele. One blood donor presented the T646A and the G829A mutations in homozygosity. It was concluded that the Ag blood group is very heterogeneous at the molecular level.222737

Produktionsstatistik 1916. Stat. Medd. IV. 55, III. Udgivet af Det statistiske Departement. Kjøbenhavn 1918.

BACKGROUND: Gamma irradiation of RBCs results in the production of reactive oxygen capable of initiating the process of membrane lipid peroxidation and accelerates the leakage of potassium ions from RBCs, resulting in an increase of internal viscosity. STUDY DESIGN AND METHODS: The elastic properties of irradiated and stored RBC units were studied using laser optical tweezers. The laser trapped the cell and the membrane elasticity was analyzed, measuring the cell deformation in six different drag velocities. Five RBC units were split into two portions. One portion received a gamma irradiation dose of 25 Gy, and the second one was used as a control and was not irradiated. All units were stored (4degreesC), and the elasticity was examined on Days 1, 14, 21, and 28. RESULTS: Elastic properties (p) from irradiated RBCs stored for 21 and 28 days were significantly affected compared with control cells (21 days: control, 0.3 +/- 0.03 x 10(-3); irradiated, 3.5 +/- 1.3 x 10(-3) dyn/cm; p < 0.001; and 28 days: control, 0.5 +/- 0.09 x 10(-3); irradiated, 14 +/- 3.2 x 10(-3) dyn/cm; p < 0.001). CONCLUSION: The sensitivity of the laser optical tweezers method showed that there is no significant change in elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not. However, beyond 21 days of storage, irradiated units demonstrate decreased elasticity.4291196119

ABO blood group in Amerindians from Brazilian Amazon

Background: The Parakana is a group of Indians with cultural similarities to the extinct Tupi group. They are an isolated native population from East Brazilian Amazon. A number of different O alleles have been found at the blood group ABO locus in populations of several ethnic origins (Caucasians, Blacks, Amerindians). Aim: The present study describes the ABO blood group polymorphism gene of the Parakana Indians. The Amerindian group was carefully selected for racial background. Subject and methods: The blood group polymorphism was analysed in genomic DNA from 62 Parakana Indians. We determined the 261G deletion, the T646A and C771T mutations described in O-1variant and the G542A substitution, using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Results: All Amerindians studied were homozygous for the 261G deletion. The frequencies of the T646A and C771T mutations in Parakanas (0.65) were lower than that observed in Kayapo, Yanomama and Arara Indians (0.91) (chi(2) = 18.24; p-nu<0.001. The G542A substitution in Parakanas was also lower (0.22) than in other tribes (0.42) (&chi;(2)=9.73; p-&nu;=0.001). Conclusions: The different O alleles including the G542A mutation are not distributed homogeneously among all Amazonian Amerindians. Our results are in agreement with other genetic markers studied previously in Parakana Indians, whose distinct genetic pattern differs from Europeans and even from other Amerindians.30222022

Measuring electrical and mechanical properties of red blood cells with double optical tweezers

Red blood cell (RBC) aggregation in the blood stream is prevented by the zeta potential created by its negatively charged membrane. There are techniques, however, to decrease the zeta potential and allow cell agglutination, which are the basis of most of antigen-antibody tests used in immunohematology. We propose the use of optical tweezers to measure membrane viscosity, adhesion, zeta potential, and the double layer thickness of charges (DLT) formed around the cell in an electrolytic solution. For the membrane viscosity experiment, we trap a bead attached to RBCs and measure the force to slide one RBC over the other as a function of the velocity. Adhesion is quantified by displacing two RBCs apart until disagglutination. The DLT is measured using the force on the bead attached to a single RBC in response to an applied voltage. The zeta potential is obtained by measuring the terminal velocity after releasing the RBC from the trap at the last applied voltage. We believe that the methodology proposed here can provide information about agglutination, help to improve the tests usually performed in transfusion services, and be applied for zeta potential measurements in other samples. (C) 2008 Society of Photo-Optical Instrumentation Engineers.13

Elastic properties of stored red blood cells from sickle trait donor units

Background and Objectives Red blood cells (RBCs) from patients with sickle cell disease present reduced deformability. The aim of this study was to analyse the elasticity of stored RBCs from patients with the sickle cell trait ( AS). Materials and Methods The cell elasticity was studied, using laser optical tweezers, on storage days 1, 14, 21, 28 and 35. Results The elasticity of RBC from AS units stored for 1, 14 and 21 days was significantly greater compared with that of control RBC cells stored for the same time-period. More than 30% of the cells from AS units stored for 28 or 35 days were very rigid and escaped from the optical trap. Conclusions RBCs became rigid during storage, suggesting that haemoglobin S might compromise the cell elasticity.85321321