Association among ACE, ESR1 polymorphisms and preeclampsia in Brazilian pregnant women.

BACKGROUND: Genetic, immune and environmental factors are involved in preeclampsia (PE) etiopathogenesis. Considering that hypertension and poor placental perfusion are important features in PE, polymorphisms in the angiotensin-converting enzyme (ACE) and estrogen nuclear receptor 1 (ESR1) genes could be involved in the predisposition and/or development of the disease. The aim of this study was to evaluate if polymorphisms in ACE and ESR1 genes were associated with PE occurrence. MATERIAL AND METHODS: This case-control study included 209 Brazilian pregnant women (107 with severe PE and 102 normotensive controls). The polymorphisms were investigated by polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis. RESULTS: No significant difference between PE versus normotensive pregnant women, as well as early versus late PE, was observed when compared the allelic and genotypic frequencies of insertion/deletion polymorphism in intron 16 of the ACE gene and the single nucleotide polymorphisms (SNPs - rs2234693 and rs9340799) of the ESR1 gene. CONCLUSION: This pioneer study involving Brazilian women showed no association among the studied polymorphisms and PE, which suggests that ins/del ACE and SNPs ESR1 do not contribute to this disease occurrence in Brazil.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis.

BACKGROUND: Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE: This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS: A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS: Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS: The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Polymorphisms in endothelial nitric oxide synthase gene in early and late severe preeclampsia.

Preeclampsia (PE) is characterized by hypertension and proteinuria, occurring after the 20th week of pregnancy in women who have had no previous symptoms. The disease progresses with generalized vasoconstriction and endothelial dysfunction. Clinically, it is important to diagnose the severe form of the disease (sPE), in which blood pressure and proteinuria are much higher. Recently, the gestational age (GA) of the onset of PE has led to the classification of this disease as early (GA <34 weeks) and late (GA ≥34 weeks). Several genetic polymorphisms affecting endothelial nitric oxide synthase (eNOS) levels or function were described, including G894T (Glu298Asp), VNTR b/a (variable-number 27-bp tandem repeat) and T-786C (promoter) polymorphisms. Thus, the aim of this study was to compare the distribution of G894T, VNTR b/a and T-786C polymorphisms and their haplotypes in Brazilian early and late sPE, as well as in normotensive pregnant. A total of 201 women were evaluated, 53 with early sPE, 45 with late sPE and 103 as normotensive pregnant women. The frequency of 894T allele was higher in late sPE vs normotensive pregnant, and 894TT genotype was higher in late sPE vs early sPE and normotensive pregnant. For VNTR b/a polymorphism, higher frequencies of aa genotype and a allele were observed in early sPE vs late sPE and normotensive pregnant. Besides, the frequency of haplotype T-b-C was higher in late sPE vs early sPE and normotensive pregnant. Considering the results found for eNOS polymorphisms, it is possible to suggest that the functional alterations induced by these two polymorphisms may influence the time of severe PE onset, although both alterations are putatively associated with low NO bioavailability. However, other studies are necessary to validate these findings and clarify this issue.

Early and late-onset preeclampsia: effects of DDAH2 polymorphisms on ADMA levels and association with DDAH2 haplotypes.

Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.

Assessment of L-arginine asymmetric 1 dimethyl (ADMA) in early-onset and late-onset (severe) preeclampsia.

Preeclampsia (PE) is characterized by hypertension and proteinuria. It has been classified in early or late according to gestational age at the onset of disease. Endothelial dysfunction plays a crucial part in its pathogenesis. NO is a potent vasodilator and ADMA is its endogenous inhibitor. We have assessed maternal ADMA levels. ADMA were increased in early [0.66 µmol/L] versus late sPE [0.47 µmol/L] (P=0.001) and versus normotensive pregnant [0.48 µmol/L] (P=0.001). Our findings suggest that high ADMA levels in early sPE could compromise NO synthesis contributing to endothelial dysfunction, leading to impaired placentation and the onset of this disease.

Neuroserpin: A potential biomarker for early-onset severe preeclampsia.

Preeclampsia is a hypertensive disease of pregnancy associated with intense inflammatory and pro-coagulant responses. Neuroserpin is a serine protease inhibitor that has been involved in neurological and immune processes and has not yet been investigated in preeclampsia. Herein, we evaluated neuroserpin levels in association with other inflammatory mediators (IL-17A, IL-33, and CXCL-16) during severe preeclampsia. The mediators' plasma levels were measured by immunoassays in 24 pregnant women with severe preeclampsia (early preeclampsia: N = 17, late preeclampsia: N = 7), 34 normotensive pregnant women, and 32 non-pregnant women. In general, pregnancy was associated with higher levels of neuroserpin, IL-17A, IL-33, and CXCL-16 than the non-pregnant state. However, this increase was attenuated in pregnancies complicated by severe preeclampsia. Although neuroserpin levels did not differ between normotensive pregnant women and pregnant women with severe preeclampsia, neuroserpin levels tended to be lower in early-onset than in late-onset severe preeclampsia. There were positive correlations between neuroserpin and IL-17A, neuroserpin and CXCL-16, and IL-17A and CXCL-16 levels in women with severe preeclampsia. In addition, although the risk for developing severe preeclampsia was higher in older women in this study, maternal age did not significantly influence the mediators' levels, nor their correlations in the preeclampsia group. In summary, our data suggest that neuroserpin might be a potential biomarker for early-onset severe preeclampsia and, that the imbalance among neuroserpin, IL-17A, IL-33, and CXCL-16 levels may be associated with the pathogenesis of preeclampsia, regardless of the maternal age.

Panoramic snapshot of serum soluble mediator interplay in pregnant women with convalescent COVID-19: an exploratory study.

Introduction: SARS-CoV-2 infection during pregnancy can induce changes in the maternal immune response, with effects on pregnancy outcome and offspring. This is a cross-sectional observational study designed to characterize the immunological status of pregnant women with convalescent COVID-19 at distinct pregnancy trimesters. The study focused on providing a clear snapshot of the interplay among serum soluble mediators. Methods: A sample of 141 pregnant women from all prenatal periods (1st, 2nd and 3rd trimesters) comprised patients with convalescent SARS-CoV-2 infection at 3-20 weeks after symptoms onset (COVID, n=89) and a control group of pre-pandemic non-infected pregnant women (HC, n=52). Chemokine, pro-inflammatory/regulatory cytokine and growth factor levels were quantified by a high-throughput microbeads array. Results: In the HC group, most serum soluble mediators progressively decreased towards the 2nd and 3rd trimesters of pregnancy, while higher chemokine, cytokine and growth factor levels were observed in the COVID patient group. Serum soluble mediator signatures and heatmap analysis pointed out that the major increase observed in the COVID group related to pro-inflammatory cytokines (IL-6, TNF-α, IL-12, IFN-γ and IL-17). A larger set of biomarkers displayed an increased COVID/HC ratio towards the 2nd (3x increase) and the 3rd (3x to 15x increase) trimesters. Integrative network analysis demonstrated that HC pregnancy evolves with decreasing connectivity between pairs of serum soluble mediators towards the 3rd trimester. Although the COVID group exhibited a similar profile, the number of connections was remarkably lower throughout the pregnancy. Meanwhile, IL-1Ra, IL-10 and GM-CSF presented a preserved number of correlations (≥5 strong correlations in HC and COVID), IL-17, FGF-basic and VEGF lost connectivity throughout the pregnancy. IL-6 and CXCL8 were included in a set of acquired attributes, named COVID-selective (≥5 strong correlations in COVID and <5 in HC) observed at the 3rd pregnancy trimester. Discussion and conclusion: From an overall perspective, a pronounced increase in serum levels of soluble mediators with decreased network interplay between them demonstrated an imbalanced immune response in convalescent COVID-19 infection during pregnancy that may contribute to the management of, or indeed recovery from, late complications in the post-symptomatic phase of the SARS-CoV-2 infection in pregnant women.

Preeclampsia: the role of tissue factor and tissue factor pathway inhibitor.

Preeclampsia (PE) is a multi-system disorder of human pregnancy, whose etiology remains poorly understood. Preeclamptic women are known to have an increased hypercoagulable state that result in excess fibrin deposition in several organs, which compromises their function. Tissue factor (TF) is the main physiological initiator of blood coagulation and its activity is regulated by a specific inhibitor known as Tissue factor pathway inhibitor (TFPI). Based on the important role of TF and TFPI in hemostasis, we hypothesize that their levels may change in the severe PE contributing to exacerbate hypercoagulable state. Some studies have assessed the balance between TF and TFPI in preeclamptic women, but results are inconsistent. Therefore, the aim of this study was to examine these inconsistencies and to assess TF and TFPI plasma levels in three groups of age matched women; pregnant with severe PE (n = 60), normotensive pregnant (n = 50) and normotensive non-pregnant women (n = 50). There was not significantly different among the three groups for TF plasma levels; severe PE women: 338.4 pg/mL (248.1-457.6), normotensive pregnant women: 301.5 pg/mL (216.4-442.9) and normotensive non-pregnant women 393 pg/mL (310.3-522.9). TFPI plasma levels were higher in severe PE comparing to normotensive pregnant women and normotensive non-pregnant women, 115.8 ng/mL (75-149.8); 80.3 ng/mL (59.6-99.7) and 74.5 ng/mL (47.1-98.0), respectively No difference was found between normotensive pregnant women and normotensive non-pregnant women. As for gestational age, a significant difference in TFPI levels was found between severe PE and normotensive pregnant women up to the 33rd week of pregnancy (p = 0.001), and severe PE and non-pregnant women up to the 34th (p = 0.01). In summary, our results indicated that TF plasma levels did not vary in the studied groups, while TFPI plasma levels were significantly increased in severe PE compared to normotensive pregnant and normotensive non-pregnant women. So, our data do not explain the exacerbated hypercoagulability state observed in severe PE. Further studies evaluating genes expression, TF activity and antigen, total and free TFPI and TFPI-2, both in plasma and obstetric tissues, throughout the pregnancy in PE (mild and severe forms) are required.

Chikungunya E2 Protein Produced in E. coli and HEK293-T Cells-Comparison of Their Performances in ELISA.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)-a prokaryotic system unable to produce post-translational modifications-or in HEK-293T mammalian cells-a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays.

Undetected Chikungunya virus co-infections in a Brazilian region presenting hyper-endemic circulation of Dengue and Zika.

BACKGROUND: Chikungunya virus (CHIKV) causes a disease characterized by acute onset of fever accompanied by arthralgia. Clinical similarities and co-circulation of other arboviruses such as Dengue virus (DENV) and Zika virus (ZIKV), have complicated their differentiation, making their diagnoses a challenge for the health authorities. Misdiagnosis is a serious issue to the management of patients and development of public health measures. OBJECTIVES: We carried out further screening of CHIKV, DENV and ZIKV cases in Minas Gerais, Brazil, after diagnostics were already issued by a state laboratory and according to the Brazilian Ministry of Health (BMH) policy. Our aim was to look for possible co-infections or previous arboviruses' exposure. STUDY DESIGN: Sera from 193 patients with symptoms of arboviral infections were tested for DEV, ZKV and/or CHIKV by the State laboratory, according to clinical suspicion and following standard BMH guidelines. After an official diagnosis was issued for each patient, we retested samples applying a broader panel of ELISA-based serological tests. RESULTS: We identified 13 patients with concurrent or consecutive infections (IgM positive for more than one arbovirus), including 11 individuals that were positive for CHIKV and other previously confirmed arbovirus infection. DISCUSSION: Guidelines established in many arbovirus-endemic countries prioritizes the diagnosis of Zika and Dengue and no further analyzes are done when samples are positive for those viruses. As a result, possible cases of co-infections with chikungunya are neglected, which affects the epidemiological assessments of virus circulation, patient management, and the development of public health policies.

Development of an enzyme-linked immunosorbent assay using recombinant protein antigen for the diagnosis of Chikungunya virus.

We describe here the development of an in-house enzyme linked immunosorbent assay (ELISA) for the diagnostic of Chikungunya virus (CHIKV) infections using a recombinant protein from CHIKV. The recombinant protein gene was designed based on 154 sequences and we used computational methods to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKVp) was synthetized and expressed in prokaryotic system. Subsequently, the protein was purified by affinity chromatography and used as antigen in an indirect ELISA. We present data regarding the optimization of the recombinant antigen production and preparation of the ELISA to detect IgG against CHIKV in human sera.

Early and late-onset preeclampsia: effects of DDAH2 polymorphisms on ADMA levels and association with DDAH2 haplotypes

Abstract Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.

Oxidative stress markers and thrombomodulin plasma levels in women with early and late severe preeclampsia.

BACKGROUND: Preeclampsia (PE) is a pregnancy disease associated with oxidative stress and endothelial dysfunction. It can be classified according to the severity and onset-time of clinical symptoms (early PE:<34 weeks, late PE:≥34 weeks). METHODS: We evaluated markers of oxidative stress (thiobarbituric acid reactive substances-TBARs and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-MTT) and endothelial lesion (thrombomodulin-TM) in early (N = 24) and late severe PE(N = 22) and normotensive pregnant women(N = 26). RESULTS: MTT levels were higher in early sPE than in normotensive pregnancy (P = 0.03). No difference was found comparing late sPE versus normotensive pregnancy, and early sPE versus late sPE. TM levels were higher in early sPE comparing to late sPE women (P = 0.05), but no difference was found between early or late sPE versus normotensive groups. TBARs levels did not differ significantly among the three groups. These data suggest that endothelial lesion and the antioxidant status are more pronounced in early sPE. Moreover, lipid peroxidation might be an early event in PE, stimulating a compensatory antioxidant defense later in pregnancy. CONCLUSIONS: Longitudinal studies involving pregnant women with risk factors for PE development and including other methods for oxidative stress and endothelial lesion determination should be conducted in order to better evaluate the role of these processes in PE pathogenesis.

Inherited thrombophilias and pre-eclampsia in Brazilian women.

OBJECTIVE: The aim of the present study was to compare the distribution of G1691A, G20210A and C677T mutations in pre-eclamptic Brazilian women and in matched control women with an uncomplicated normal pregnancy. STUDY DESIGN: these mutations were investigated by PCR-RFLP in 83 normal pregnancies (control group) and in 30 pre-eclamptic pregnant women (severe form). RESULTS: G1691A mutation was detected neither in the control group nor in pre-eclamsia women. G20210A mutation was detected in heterozygosis in 3 (3.61%) control subjects, but not in pre-eclampsia group. C677T mutation was detected in homozygosis in 6 (7.23%) control subjects and 2 (6.67%) pre-eclamptic women and in heterozygosis in 31 (37.3%) control subjects and 12 (40%) pre-eclamptic women. Differences in the mutation frequencies detected in the two groups were not statistically significant. CONCLUSION: No correlation was observed between pre-eclampsia and presence of G1691A, G20210A and C677T mutations in Brazilian women.

Hypercoagulability markers in young asymptomatic heterozygous carriers of factor V Leiden (G1691A) or prothrombin (G20210A) variant.

BACKGROUND: Mutations in factor V (factor V Leiden-G1691A) and prothrombin (G20210A) genes are important risk factors for thrombophilia due to their high incidence in patients with thromboembolic events, especially among the young. However, it is not clear if levels of hypercoagulability markers are significantly altered in asymptomatic young carriers of factor V Leiden or prothrombin G20210A. METHODS: Hemostatic status of 32 asymptomatic young individuals carrying these mutations and of 18 normal control individuals was investigated through the determination of plasma thrombomodulin (TM), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and D-dimer. RESULTS: No significant differences were observed in these hemostatic markers when comparing groups of individuals carrying mutations and the control group. CONCLUSION: Analysis of these results leads to the conclusion that the presence of these mutations, in the absence of acquired risk factors, does not constantly predispose these young carriers to a state of hypercoagulability.

The unexpected beneficial role of smoking in preeclampsia.

We have recently investigated the association between the risk of developing PE and clinical, hemostatic, inflammatory and genetic parameters of 108 severe preeclamptic women. A multivariate logistic regression analysis was performed to assess what variables are independent risk factors for PE. Univariate analysis was performed including the variables in age, smoking condition, multiple pregnancy, blood group, phenotypes and alleles of IL-4, IL-5, IL-10, IL-6, IL-8, IL-12, IL-1ß, IFN-γ, TNF-α, and the plasma levels of FVII, FVIIa, FVIIa-AT, FVIII, FVW, ADAMTS13, D-Di, PAI-1, ADMA. Those variables whose P<0.20 (smoking, multiple pregnancy, blood group, phenotype IL-6, IFN-γ allele, IL-10 allele and FVII) were selected to the multivariate logistic regression. In the final model, only FVII, IFN-γ allele and smoking were independently associated to severe PE (P<0.0001, P<0.0001 and P=0.008, respectively). Increased FVII plasma levels and IFN-γ "T" allele were associated to an increased probability of developing PE (OR 1.001, 95% CI [1.001-1.002], and OR 4.81 95% CI [2.42-9.60], respectively). On the other hand, smoker status was associated with a 4.72 decreased chance of PE occurrence (OR 4.72, 95% CI [1.51-14.75]). In this article we also reviewed the studies that investigate the risk of PE in pregnant women who smoked, as well as the effect of tobacco extract in cells or animal models. The hypotheses proposed to explain the biological mechanism by which smoking during pregnancy reduces the risk of PE was also discussed.

Increased Levels of sENG and sVCAM-1 and Decreased Levels of VEGF in Severe Preeclampsia.

BACKGROUND: Preeclampsia (PE) is characterized by hypertension and proteinuria after the 20th week in pregnant women who have had no previous symptoms. Clinically, it is important to diagnose the severe form of the disease, in which blood pressure is much higher. Imbalance between angiogenic and antiangiogenic factors, as well as changes in adhesion molecules seem to contribute to the endothelial dysfunction and PE clinical manifestations. The aim of this study was to assess plasma levels of the angiogenic factors (free vascular endothelial growth factor (VEGF) and soluble endoglin (sEng)) and adhesion molecules (soluble forms of intercellular adhesion molecule-1 (sICAM-1) and soluble forms of vascular cell adhesion molecule-1 (sVCAM-1)) in severe PE (sPE), in order to clarify the circulating profile of these factors. METHODS: Sixty women with sPE (34 with early sPE and 26 with late sPE), and 60 normotensive pregnant were enrolled in this study. Free VEGF, sICAM-1, sVCAM-1, and sEng plasma levels were determined by ELISA. RESULTS: Increased sEng and sVCAM-1 and decreased free VEGF plasma levels were found in women with sPE, compared with normotensive pregnant group. However, no significant difference was observed comparing early and late sPE. CONCLUSION: Our data confirm the imbalance in changes in angiogenic and antiangiogenic factors, as well changes in adhesion molecule (sVCAM-1) in PE. These findings give support to the hypothesis that circulating angiogenic proteins and endothelial dysfunction may have an important biologic role in PE. Data from prospective, longitudinal studies producing serial determinations of these molecules throughout pregnancy are needed to better understanding the relevance of these markers in PE diagnosis and prognosis.

Parvovirus B19 (B19) and cytomegalovirus (CMV) infections and anti-erythropoietin (anti-EPO) antibodies in patients on dialysis hyporesponsive to erythropoietin therapy.

BACKGROUND: Approximately 10% of patients receiving recombinant human erythropoietin (rHuEPO) do not respond to the treatment. We evaluated parvovirus B19 (B19) and cytomegalovirus (CMV) infections and antierythropoietin (anti-EPO) antibodies as potential causes of anemia in dialyzed patients, hyporesponsive to rHuEPO. METHODS: Data from 120 dialyzed patients, receiving rHuEPO alfa, were collected: demographic characteristics, rHuEPO dose, duration of rHuEPO treatment and time on dialysis, etiology of chronic kidney disease and transfusion history. Serology and PCR were performed to address B19 and CMV infection status. An ELISA was developed to detect anti-EPO antibodies. RESULTS: rHuEPO resistance correlated with high ferritin levels (p = 0.001) and short time on dialysis (p = 0.012). B19 DNA was found in 10 (8.3%) dialyzed patients and CMV DNA was detected in 33 (27.5%). There was no significant correlation between B19 infection and anemia,while a tendency of correlation between active CMV infection and hemoglobin levels or hematocrit value (p= 0.069 and p= 0.070, respectively) has been observed. Anti-EPO antibodies were not detected in any patient. CONCLUSIONS: B19 infection is a rare complication in dialyzed patients and should be investigated after exclusion of other common causes, while CMV infection is rather common. The role of CMV infection in the hyporesponsiveness in dialyzed patients should be further evaluated in other studies. Our data suggest that anti-EPO antibodies are not involved in rHuEPO resistance in this population.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.