scholarly journals Development and Validation of a PCR-ELISA for the Diagnosis of Symptomatic and Asymptomatic Infection byLeishmania (Leishmania) infantum

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Fernanda Alvarenga Cardoso Medeiros ◽  
Luciana Inácia Gomes ◽  
Edward Oliveira ◽  
Carolina Senra Alves de Souza ◽  
Maria Vitória Mourão ◽  
...  
Keyword(s):  
Detection Limit ◽  
Peripheral Blood ◽  
Leishmania Infantum ◽  
Asymptomatic Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Individuals ◽  
Blood Samples ◽  
Hiv Coinfection ◽  
Satisfactory Precision ◽  
Asymptomatic Individuals

AkDNAPCR enzyme-linked immunosorbent assay (kDNAPCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of thekDNAPCR-ELISA were defined in a proof-of-concept phase. A reference strain ofLeishmania (Leishmania) infantumand a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients withLeishmania/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. ThekDNAPCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA fromL.(L.) infantumand 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1–100%), and the specificity was 95% (95% CI: 83.5–98.6%). ThekDNAPCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions.

Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis

Parasitology ◽  
2020 ◽  
pp. 1-5
Author(s):  
Loren Queli Pereira ◽  
Márcia Maria Ferreira-Silva ◽  
Cristhianne Molinero Andrade Ratkevicius ◽  
César Gómez-Hérnandez ◽  
Fernanda Bernadelli De Vito ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Infantum ◽  
Blood Donors ◽  
Asymptomatic Infection ◽  
Molecular Techniques ◽  
Enzyme Linked Immunosorbent Assay ◽  
Conventional Pcr ◽  
Asymptomatic Patients ◽  
Polymerase Chain ◽  
Asymptomatic Individuals

Abstract Visceral leishmaniasis is an endemic protozoonosis observed in over 60 countries, with over 500 000 new cases recorded annually. Although the diagnostic procedure of its symptomatic forms is well established, for asymptomatic patients, who represent about 85% of those infected, there is no consensus on the best method for its identification. Recent studies have presented molecular techniques as viable identification methods, with good sensitivity and specificity indices in asymptomatic individuals. Therefore, we aimed to use molecular methods to assess their effectiveness in identifying the presence of asymptomatic infection by Leishmania infantum (L. infantum) individuals from endemic regions of Brazil. Screening was performed by real-time polymerase chain reaction (qPCR) and confirmed by sequencing the cytochrome B gene. Of the 127 samples [from 608 blood donors who had participated in a previous study, of which 34 were positive by the enzyme-linked immunosorbent assay (ELISA) rK39] tested by qPCR, 31 (24.4%) were positive. In the sequencing of 10 qPCR-positive samples, five were identified as L. infantum. Complimentary samples of the ELISA rK39 and conventional PCR showed only reasonable and low agreement with qPCR, respectively. The qPCR confirmed the presence of infection in five of the 10 sequenced samples, ELISA confirmed three, and the conventional PCR confirmed none.

scholarly journals Circulating Natural IgM Antibodies against Angiogenin in the Peripheral Blood Sera of Patients with Osteosarcoma as Candidate Biomarkers and Reporters of Tumorigenesis

2010 ◽  
Vol 2 ◽  
pp. BIC.S6040 ◽  
Author(s):  
Yulia A. Savitskaya ◽  
Genaro Rico ◽  
Luis Linares ◽  
Roberto González ◽  
René Téllez ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Sensitivity And Specificity ◽  
Peripheral Blood ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Healthy Individuals ◽  
Igm Antibodies ◽  
Potential Biomarker ◽  
Increased Risk ◽  
Natural Igm

Background Tumor immunology research has led to the identification of a number of tumor-associated self antigens, suggesting that most tumors trigger an immunogenic response, as is the case in osteosarcoma, where the detection of natural serum IgM antibodies might achieve the diagnosis of osteosarcoma. Natural IgM antibodies to tumor-associated proteins may expand the number of available tumor biomarkers for osteosarcoma and may be used together in a serum profile to enhance test sensitivity and specificity. Natural IgM antibodies can be consistently detected in the peripheral blood sera months to years before the tumor is diagnosed clinically. The study of the level of a potential biomarker many months (or years) prior to diagnosis is fundamentally important. Integrated circulating and imaging markers in clinical practice treating osteosarcoma have potential applications for controlling tumor angiogenesis. Objectives To study the expression of natural IgM antibodies to the tumor antigens of angiogenesis in the peripheral blood sera of osteosarcoma patients and healthy individuals, and to develop serum-based predictive biomarkers. Methods Peripheral venous blood samples were collected from 117 osteosarcoma patients and 117 patients with other tumors. All diagnosis was histologically confirmed. Staging of patients was performed according to the Enneking Surgical Staging System. The control group consisted of 117 age- and sex- matched healthy individuals. In this study, novel immunoconjugates were designed, synthesized and then used to develop a rapid, specific and sensitive enzyme-linked immunosorbent assay (ELISA) method to detect angiogenin (ANG)–IgM directly in the peripheral blood sera of humans. Results Serum ANG–IgM levels are significantly higher in osteosarcoma patients than in healthy individuals ( P < 0.005). Serum ANG–IgM levels varied widely, but were highly dependent on the concentration of IgM (r = 0.85; P < 0.0005). We found ANG–IgM in the sera of 85% of newly diagnosed osteosarcoma patients and ANG–IgM levels were significantly higher in osteosarcoma patients compared to any other tumors ( P < 0.001). Conclusions These results demonstrated that the combined biomarker ANG–IgM has greater sensitivity and specificity in early diagnosis of osteosarcoma patients than the traditional biomarkers (ANG and vascular endothelial growth factor). Circulating ANG–IgM immune complexes can potentially serve as a biomarker for increased risk of osteosarcoma, because relatively high serum levels were also detected in otherwise healthy individuals with a first degree family history of osteosarcoma and in patients with a diagnosis of benign conditions. Immunological aspects of angiogenesis for managing osteosarcoma will have a practical value in early diagnosis, prognosis and monitoring response to antiangiogenic therapy.

scholarly journals SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples

2014 ◽  
Vol 40 (1) ◽  
pp. 81-87 ◽  
Author(s):  
Mehrdad Ghasemian ◽  
Mohammad Javad Gharavi ◽  
Lame Akhlaghi ◽  
Mehdi Mohebali ◽  
Ahmad Reza Meamar ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Leishmania Infantum ◽  
Sybr Green ◽  
Blood Samples

Correlation of levels of extracellular vesicles in peripheral and pulmonary blood plasma with pathological stages of lung cancer patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15558-e15558
Author(s):  
Hyun Koo Kim ◽  
Byeong Hyeon Choi ◽  
Yu Hua Quan ◽  
Jiyun Rho ◽  
Sunghoi Hong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Patients ◽  
Peripheral Blood ◽  
Human Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Individuals ◽  
Peripheral Vein ◽  
Healthy Human ◽  
Lung Cancer Patients ◽  
Pathological Stages

e15558 Background: Exosome concentration is known to be higher in cancer patients than in healthy individuals. In this study, we observed that the levels of exosomes differ in tumor-draining pulmonary blood and in peripheral blood in animal models and human subjects at different pathological stages of lung cancer. Methods: Ten rabbits and 40 humans formed the study cohorts. Blood was collected from a peripheral vein in all groups, and pulmonary blood was collected intraoperatively from all groups, except the healthy human controls. Quantitative analysis of exosomes was performed by nanoparticle tracking assay, CD63 enzyme-linked immunosorbent assay, and western blotting. Results: The peripheral blood of lung cancer-bearing animals and patients with lung cancer carried higher amounts of exosome than that from healthy controls ( p < 0.01 and p < 0.001, respectively). Moreover, pulmonary blood from lung cancer-bearing animals and patients had significantly higher exosome levels, compared to preoperative peripheral blood ( p < 0.01 and p < 0.0001, respectively). In patients, pulmonary exosome levels showed higher correlation with pathological stages of lung cancer than the peripheral exosome levels. Conclusions: Exosome levels increased with increasing grade of lung cancer, and this trend was more prominent in the pulmonary than in the peripheral blood.

Reduced Proteasomal Activity in Marrow Cells From Myelodysplastic Syndrome (MDS) Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1776-1776
Author(s):  
Stela Karabina ◽  
Eleni Thanopoulou ◽  
Sotirios D Athanasiou ◽  
Argiris Symeonidis ◽  
Nicholas Zoumbos
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Proteasome Activity ◽  
Healthy Individuals ◽  
Blood Samples ◽  
Proteasomal Activity ◽  
Healthy Donors ◽  
Intracellular Proteins ◽  
Blood Mononuclear Cells ◽  
Marrow Cells

Abstract Abstract 1776 Poster Board I-802 Introduction The proteasome is a multicatalytic complex that is responsible for the degradation of many intracellular proteins in the cytoplasm and nucleus of all eukaryotic cells. The chymotryptic-like (ChT-L) activity is one of the three proteasomal activities and is rate-limiting in the degradation of intracellular proteins. Such proteins include natural unfolded proteins, oxidized, mutated or damaged proteins and many short–lived proteins such as cell cycle proteins. The correlation between MDS and the presence of increased oxidative stress as well as oxidized proteins within the cell has already been reported. Insufficient proteasomal activity causes the accumulation of oxidatively modified proteins something that may lead to the misregulation of cellular homeostasis. In addition, its role to the pathogenesis of many diseases has already been documented and many studies over the past years strongly indicate the implication of proteasome in many hematological malignancies. Patients and methods: In this study we examined the proteasomal activity of mononuclear cells and immunomagnetically isolated CD34+ marrow cells from 24 marrow and 9 peripheral blood samples from MDS patients (10 RA, 2 RARS, 5 RCMD, 10 RAEB I, 2 RAEB II, 4 CMML) compared to the cells from 6 marrow samples of age matched healthy individuals, 8 mobilized peripheral blood samples (mPB) and 14 blood samples from healthy donors. For each sample, cells were lysed with Dithiothreitol (DTT) followed by incubation with the fluorescent conjugated peptide Suc-LLVY-AMC. The fluorescence intensity was directly dependent to ChT-L activity within the cells. The proteasome inhibitor MG-132 was used as negative control. We also examined the amount of the β1 subunit of the proteasome in marrow mononuclear cells from MDS patients, by flow cytometry. Results Proteasome activity was significantly reduced in MDS marrow mononuclear cells compared with the cells from healthy individuals. The reduction was 55% and 75% compared with mononuclear cells from marrow samples of healthy donors and mPB samples, respectively. The activity was similarly reduced in both CD34+ and CD34- marrow cells from MDS patients compared to the cells from healthy individuals (70% and 60% reduction respectively). Further analysis showed high variation of the amount of the β1 proteasomal subunit in MDS patients contrary to healthy individuals. Additionally, cells from the mPB samples possessed higher levels of proteasomal activity and β1 subunit compared to the cells from healthy donors. Proteasome activity of blood mononuclear cells from MDS patients was similar to the cells from the blood samples of healthy individuals although the variance was high in both study groups. Conclusions Both mononuclear and progenitor CD34+ marrow cells from MDS patients have reduced proteasomal activity compared to the same cell populations from healthy individuals. However, no difference in activity was detected between MDS and healthy individuals blood mononuclear cells. The quantitative assessment of the proteasome shows high variation within the MDS group and further investigation is required. Disclosures No relevant conflicts of interest to declare.

scholarly journals Feline Leukemia Virus Detection by ELISA and PCR in Peripheral Blood from 68 Cats with High, Moderate, or Low Suspicion of having FeLV-Related Disease

1996 ◽  
Vol 8 (1) ◽  
pp. 25-30 ◽  
Author(s):  
Marion L. Jackson ◽  
Deborah M. Haines ◽  
Susan M. Taylor ◽  
Vikram Misra
Keyword(s):  
Relative Risk ◽  
Peripheral Blood ◽  
Leukemia Virus ◽  
Feline Leukemia Virus ◽  
Positive Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blood Samples ◽  
Related Disease ◽  
Significant Difference ◽  
V Antigen

Clinicopathologic criteria were used to group 68 cats according to high, moderate, or low suspicion of having feline leukemia virus (FeL V)-related disease. Peripheral blood samples were tested for FeL V antigen by enzyme-linked immunosorbent assay (ELISA) and for FeL V DNA by polymerase chain reaction (PCR). There was no significant difference between ELISA and PCR results in the 68 cats. In the high-suspicion group, 46% (11/24) of cytopenic cats were test positive (ELISA and PCR) and 87% (13/15) with hemopoietic neoplasms were test-positive. Also within the high suspicion group, test-positive cats were 2.5 times more likely to die within the 1 year follow-up period than were test-negative (ELISA and PCR) cats. Among cats in the moderate-suspicion group, 15% (2/13) were test-positive, and none (0/16) of the cats in the low suspicion group was test positive. The relative risk of a positive test (ELISA and PCR) in the high suspicion group was 3.7 times that for the moderate-suspicion group and 22.8 times that for the low suspicion group. There was no significant difference in the relative risk of a positive test result between the moderate and low suspicion groups. The results indicate that FeL V detection by PCR can be adapted for diagnostic purposes using peripheral blood samples, however, results do not differ significantly from FeL V ELISA results. Also, a proportion of cats with a high suspicion of having FeL V-related cytopenia and hemopoietic tumors are negative for both circulating FeL V antigen and DNA. These cats may not have FeL V-related disease, or FeL V may exist in a disease-producing but nonreplicating form ultimately detectable by PCR in tissues other than peripheral blood.

scholarly journals Compartmentalization and Transmission of Multiple Epstein-Barr Virus Strains in Asymptomatic Carriers

2003 ◽  
Vol 77 (3) ◽  
pp. 1840-1847 ◽  
Author(s):  
Diane Sitki-Green ◽  
Mary Covington ◽  
Nancy Raab-Traub
Keyword(s):  
Oral Cavity ◽  
Peripheral Blood ◽  
Epstein Barr Virus ◽  
Asymptomatic Infection ◽  
Time Points ◽  
Barr Virus ◽  
Epstein Barr ◽  
Asymptomatic Individuals ◽  
Multiple Strains ◽  
Virus Strains

ABSTRACT Infection with the Epstein-Barr virus (EBV) is often subclinical in the presence of a healthy immune response; thus, asymptomatic infection is largely uncharacterized. This study analyzed the nature of EBV infection in 20 asymptomatic immunocompetent hosts over time through the identification of EBV strain variants in the peripheral blood and oral cavity. A heteroduplex tracking assay specific for the EBV gene LMP1 precisely identified the presence of multiple EBV strains in each subject. The strains present in the peripheral blood and oral cavity were often completely discordant, indicating the existence of distinct infections, and the strains present and their relative abundance changed considerably between time points. The possible transmission of strains between the oral cavity and peripheral blood compartments could be tracked within subjects, suggesting that reactivation in the oral cavity and subsequent reinfection of B lymphocytes that reenter the periphery contribute to the maintenance of persistence. In addition, distinct virus strains persisted in the oral cavity over many time points, suggesting an important role for epithelial cells in the maintenance of persistence. Asymptomatic individuals without tonsillar tissue, which is believed to be an important source of virus for the oral cavity, also exhibited multiple strains and a cyclic pattern of transmission between compartments. This study revealed that the majority of patients with infectious mononucleosis were infected with multiple strains of EBV that were also compartmentalized, suggesting that primary infection involves the transmission of multiple strains. Both the primary and carrier states of infection with EBV are more complex than previously thought.

scholarly journals Πρότυπο μεθυλίωσης σε ασθενείς με ιδιοπαθή φλεγμονώδη νόσο του εντέρου

2014 ◽  
Author(s):  
Παντελής Καρατζάς
Keyword(s):  
Peripheral Blood ◽  
Statistical Tests ◽  
Methylation Status ◽  
Methylation Profile ◽  
Healthy Individuals ◽  
Intestinal Tissue ◽  
Tissue Samples ◽  
Blood Samples ◽  
Number Of Patients ◽  
Show Evidence

Objective This thesis aimed to investigate the methylation profile of 22 genes associated with thepathogenesis of IBD in patients with ulcerative colitis (UC) or Crohn’s Disease (CD) andcompare them with the methylation profile of the same genes in healthy controls. Anadditional purpose of the study was to investigate whether methylation profile of these genesbetween inflamed intestinal tissue and peripheral blood are in concordance, hoping to indicatea possible future use of methylation as biomarker.Materials & MethodsIsolated peripheral blood samples and inflamed intestinal tissue samples were collectedfrom 24 patients with IBD, 12 of them suffering from UC and 12 from CD. The control groupconsisted of 12 healthy subjects without any personal and family history of digestive diseases.The promoter methylation status of genes involved in inflammation and autoimmunity wasprofiled using the Human Inflammatory Response and Autoimmunity EpiTect Methyl IISignature PCR Array profiles. Methylation was considered to be hyper-methylated if >20%according to the instructions of the manufacturer. The microarrays were validated with Quantitative Real-time PCR.The statistical analysis was performed using non-parametricstatistical tests (non-parametric statistical tests).ResultsRegarding CD, the methylation status of IL10RA, IL13, IL13RA1 and IL17C inperipheral blood samples did not differ significantly from the methylation status of healthycontrols. Only three genes – ATF2, CXCL5 and IL12B showed higher methylation in CDcompared to controls, but they did not exceed the threshold of 20% for hypermethylation. Allother genes tested appear lower methylation than controls.Regarding UC, methylation status of CXCL6 and IL13RA1 in peripheral blood samplesdid not differ significantly from the methylation status of healthy individuals. Five genes(CXCL14, CXCL5, GATA3, IL17C and IL4R,) were found to be significantly hypermethylatedin UC patients compared to healthy individuals. Some genes show higher methylation thancontrols, but they do not exceed 20% methylation threshold for hypermethylation. All other genes show lower methylation in UC compared to controls.Active cases of both CD and UC could be promptly distinguished from healthycontrols based on the signatures provided by the methylation profiles in peripheral bloodsamples. Based on these results - despite the relatively limited number of patients - it waspossible to define distinct signatures for active CD vs. active UC. Specifically, CXCL14,CXCL5, GATA3, IL17C and IL4R genes were hyper-methylated in UC compared to CD. Inaddition, CXCL6 which did not differ significantly between UC and controls appeared hypermethylatedcompared to CD; in contrast, IL13 which did not differ significantly between CDand controls appeared hypermethylated in CD compared to UC. Moreover the real-timequantitative PCR conducted for seven genes (CCL25, IL13, IL17RA, IL17A, IL12B, CXCL5,and IL4R) confirmed the causal relationship between hyper-methylation and lower expressionof genes. Finally, it was confirmed that methylation profile in intestinal tissue and peripheralblood are in concordance. ConclusionsIn this study we have identified panels of genes that show evidence of differentialmethylation between UC and controls, as well as CD and UC. Moreover there is strongevidence that methylation level in intestinal tissue samples is well related to methylation levelin whole blood samples. Our findings suggest that these genes play an important role in IBDpathogenesis, as differential methylation status observed affects gene expression at the mRNAlevel. In conclusion, the different methylation levels between CD and UC could play a role inconfirming the diagnosis of IBD, determining the exact type of IBD (UC or CD) and possiblyestimating the activity of disease

Detection of Leishmania infantum Minicircle Kinetoplast DNA in Bone Marrow and Peripheral Blood Samples of Paediatric Patients from Children`s Hospital of Tabriz Medical University

2008 ◽  
Vol 7 (2) ◽  
pp. 175-181
Author(s):  
Mohammad Ali Hossein . ◽  
Leila Mehdizadeh Fan . ◽  
Abbass Ali Hosseinpo . ◽  
Mohammad Ali Mahmood . ◽  
Safar Farajnia . ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Leishmania Infantum ◽  
Kinetoplast Dna ◽  
Blood Samples ◽  
Paediatric Patients ◽  
Medical University
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