Development of a Method to Detect Mycobacterium paratuberculosis in the Blood of Farmed Deer Using Actiphage® Rapid

Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne's disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.

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Comparison of rapid cassette test and micro ELISA method for Helicobacter pylori diagnosis in patients with gastric complaints

10.21203/rs.2.18466/v1 ◽

2019 ◽

Author(s):

Sadik Akgun ◽

Sezgin Barutcu ◽

Sumeyya Capuk ◽

Arzu Tanriverdi ◽

Serihan Kübra Emikoglu ◽

...

Keyword(s):

Helicobacter Pylori ◽

Quantitative Methods ◽

Elisa Test ◽

Diagnostic Methods ◽

Clinical Microbiology Laboratory ◽

Test Results ◽

Serum Samples ◽

Blood Samples ◽

Study Results ◽

H Pylori

Abstract Background: Helicobacter pylori (H. pylori) is a significant contributor of various gastrointestinal disorders and cancers all around the world. Its diagnosis is dependent on several qualitative and quantitative methods. The present study aims to compare the results of rapid cassette and micro ELISA test methods for diagnosis of H. pylori and determining associations with patient endoscopy reports. Methods: The study was performed using blood samples collected from 224 patients (142 (63%) females and 82 (37%) males) in various clinics between January 2018 and August 2019, which were sent to the Clinical Microbiology Laboratory of Training Hospital. Serum samples obtained after centrifugation of the blood samples were initially tested with rapid H. pylori IgG cassette method, and afterwards in the auto analyzer using ELISA assays specific for H. pylori. Results: Upper gastrointestinal system endoscopy was performed in 88 of these patients, and biopsy results confirmed definitive diagnosis of H. pylori infection in 63 of the patients. Rapid H. pylori cassette test results of the 224 patients were negative for 158 (70.5%) patients and positive for 66 (29.5%) patients, whereas micro ELISA IgA test results were negative for 110 (49.1%) patients and positive for 114 (50.9%) patients. Micro ELISA IgG test results were negative for 85 (37.9%) patients and positive for 139 (62.1%) patients. Conclusions: Invasive diagnostic methods for H. pylori infection may sometimes be inconvenient, and therefore the diagnosis may have to rely on non-invasive tests. Bases on the study results, we believe micro ELISA test results are more reliable with regard to avoidance of missed diagnosis. Keywords: Helicobacter pylori, Rapid casette test, ELISA, Gastric ulcer

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Immunophenotyping pattern characterization in canine blood: towards a clinical application

Acta Veterinaria Brno ◽

10.2754/avb201685020139 ◽

2016 ◽

Vol 85 (2) ◽

pp. 139-145

Author(s):

Leticia G. León ◽

Fatima Cruz Lopez ◽

M. Luisa Fermín ◽

Guillermo Mejías ◽

Elisabeth Kremmer ◽

...

Keyword(s):

Whole Blood ◽

Mononuclear Cells ◽

Gradient Centrifugation ◽

Blood Collection ◽

Human Beings ◽

Blood Samples ◽

Cell Counts ◽

Significant Difference ◽

Blood Lysis ◽

Whole Blood Samples

Immunophenotyping is a widely used method for a precise diagnosis and classification of haematopoietic neoplasia in human beings and also in dogs. The gold standard for cell preparation is density gradient centrifugation of mononuclear cells. Alternatively, another way to separate human leukocytes is carrying out whole blood lysis. The aim of this study was to validate whole blood lysis as an alternative method in clinical veterinary procedures using an immunophenotype panel of leukocytes designed by our group. Flow cytometry study of adult canine leukocytes subset groups, using whole blood lysis or mononuclear cells tested against an array of canine leukocyte antibodies were done. Besides differential white blood cell counts were done. Also immunophenotyping studies in whole blood samples stored at 4 °C for 48 h were performed. The Coefficient Variation values were less than 20%, for most of the comparison. Consistent results were observed in phenotyping canine peripheral blood leukocytes. Stability results indicated that whole blood samples might be stored for 48 h without a significant difference in the data compared to samples processed immediately after blood collection. This study shows that whole blood lysis represents an efficient and quick alternative for canine leukocyte preparation. In addition, samples can be analysed immediately or stored for 48 h without a significant difference between them. This is relevant for veterinary medicine considering the lack of facilities in many laboratories to process samples.

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Automated Latex Agglutination and ELISA Testing Yield Equivalent D-Dimer Results in Patients with Recent Myocardial Infarction

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614846 ◽

1999 ◽

Vol 82 (11) ◽

pp. 1412-1416 ◽

Cited By ~ 5

Author(s):

Wojciech Zareba ◽

John Horan ◽

Arthur Moss ◽

Joel Kanouse ◽

◽

...

Keyword(s):

Myocardial Infarction ◽

Elisa Test ◽

Mean Value ◽

Latex Agglutination ◽

Test Results ◽

Coronary Death ◽

Elisa Testing ◽

Strong Linear Correlation ◽

Highly Correlated ◽

D Dimer

SummaryOur previous prospective study of post-infarction patients described a strong and significant association of increased plasma D-dimer concentrations in those who experienced a subsequent coronary death or non-fatal myocardial infarction. In the present study, we compare results on stored plasma obtained two months after the index myocardial infarction from 1,038 patients of this trial, using a simple automated latex agglutination (LA) assay in parallel with the standard ELISA test. Results show a somewhat higher mean value for the LA assay (702 ± 1092 vs. 638 ± 986 ng/ml, p = 0.0002), a strong linear correlation of the two assays (r = 0.86) and 88% agreement for values below 500 ng/ml by the ELISA test. D-dimer concentrations determined by each assay were highly correlated in patients with subsequent coronary artery events (p = 0.93) and quartile values for both the LA and ELISA were equally predictive of such events (p = 0.003 and p = 0.001, respectively). This is the first demonstration that a latex agglutination assay for D-dimer can be used to assess the prognostic risk of recurrent coronary thrombotic disease after myocardial infarction

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Molecular characteristics of HBV infection among blood donors tested HBsAg reactive in a single ELISA test in southern China

BMC Infectious Diseases ◽

10.1186/s12879-020-05747-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Xianlin Ye ◽

Tong Li ◽

Ran Li ◽

Heng Liu ◽

Junpeng Zhao ◽

...

Keyword(s):

Viral Load ◽

Hepatitis B ◽

Nested Pcr ◽

Blood Donors ◽

Southern China ◽

Core Promoter ◽

Elisa Test ◽

Hbv Infection ◽

Molecular Characteristics ◽

Blood Samples

Abstract Background Hepatitis B virus (HBV) infection is a major concern for blood safety in high-prevalence HBV countries such as China. In Shenzhen, dual hepatitis B surface antigen (HBsAg) enzyme-linked immunosorbent assays (ELISAs) have been adopted in parallel with nucleic acid testing (NAT) for donors for over a decade. A small proportion of blood donors test reactive (R) for HBsAg but negative through routine NAT, which can lead to HBV infection with an extremely low viral load. Objectives We aimed to investigate and analyze the molecular characteristics of HBV among blood donors that tested HBsAg R in a single ELISA test. Methods Blood donations were evaluated in this study if confirmed HBsAg R through one of two ELISA kits. Samples with non-reactive (NR) results by NAT were collected and tested for HBsAg by chemiluminescent microparticle immunoassay (CLIA) with a neutralization test. The level of HBsAg was further assessed by electrochemiluminescence immunoassay (ECLIA). The viral basic core promoter (BCP) and pre-core (PC) and S regions were amplified by nested PCR. Quantitative real-time PCR (qPCR) for viral load determination and individual donation (ID)-NAT were adopted simultaneously. HBsAg was confirmed with CLIA, ECLIA, nested PCR, qPCR, and ID-NAT. Results Of the 100,252 donations, 38 and 41 were identified as HBsAg R with Wantai and DiaSorin ELISA kits, respectively. Seventy-nine (0.077%, 79/100,252) blood samples with ELISA R-NR and NAT NR results were enrolled in the study. Of these, 17 (21.5%,17/79) were confirmed as HBsAg-positive. Of the 14 genotyped cases, 78.6% (11/14) were genotype B, and C and D were observed in two and one sample, respectively. Mutations were found in the S gene, including Y100C, Y103I, G145R, and L175S, which can affect the detection of HBsAg. A high-frequency mutation, T1719G (93.3%), was detected in the BCP/PC region, which reduced the viral replication. Conclusion A small number of blood samples with HBsAg ELISA R-NR and NAT NR results were confirmed as HBV infection, viral nucleic acids were found in most of the samples through routine NAT methods. It is necessary to employ more sensitive and specific assays for the detection of HBV infection among blood donors.

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Comparison of capillary, venous and buffy coat blood samples in detecting Plasmodium species among malaria suspected patients attending at Hamusite health center. A cross-sectional study

BMC Infectious Diseases ◽

10.1186/s12879-021-06290-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Getu Abeje ◽

Woyneshet Gelaye ◽

Getaneh Alemu

Keyword(s):

Health Center ◽

Detection Rate ◽

Venous Blood ◽

Cross Sectional Study ◽

Buffy Coat ◽

Blood Film ◽

Capillary Blood ◽

Sectional Study ◽

Cross Sectional ◽

Blood Samples

Abstract Background Both capillary and venous blood samples have been interchangeably used for the diagnosis of malaria in Ethiopia. However, Plasmodium parasites are thought to be more concentrated in capillary than in venous blood. Hence, selecting a sample source where parasites are more concentrated is indispensable approach in order to maximize the accuracy of blood film microscopy. Therefore, the present study aimed to compare the detection rate and the parasitemia level of Plasmodium species from conventional capillary and venous blood films, and buffy coat preparations. Methods A facility based cross-sectional study was conducted from Feburary to March 2020 among 210 febrile patients attending Hamusite health center, northwest Ethiopia. Capillary and venous blood samples were collected and buffy coat was prepared from each sample. Thin and thick blood films were prepared, stained, and examined microscopically following standard protocol. Data were analysed using Statistical Package for Social Sciences Software version 20 and Med-Calc software version 19.3. Results Capillary blood buffy coat (61/210, 29.0%) had significantly higher detection rate as compared to capillary (48/210, 22.9%) and venous (42/210, 20.0%) blood films (p < 0.001). However, no significant difference was observed between capillary and venous blood films (p = 0.070) in detecting Plasmodium species. The highest and the lowest mean asexual stage parasite counts were found in capillary blood buffy coat (4692.88) and venous blood (631.43) films, respectively showing significant variations (p < 0.001). Mean gametocyte count was also highest in capillary blood buffy coat (3958.44). As compared to capillary blood buffy coat, the sensitivity of venous blood buffy coat, capillary blood film and venous blood film were 73.8, 78.7, 68.9%, respectively. Conclusion Capillary blood buffy coat samples showed the highest sensitivity in detecting and quantitating malaria parasites that its use should be promoted in clinical settings. However, conventional capillary and venous blood films could be used interchangeably.

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Evaluation of a Preclinical Blood Test for Scrapie in Sheep Using Immunocapillary Electrophoresis

Journal of AOAC International ◽

10.1093/jaoac/89.3.720 ◽

2006 ◽

Vol 89 (3) ◽

pp. 720-727 ◽

Cited By ~ 12

Author(s):

Roy Jackman ◽

David J Everest ◽

Mary Jo Schmerr ◽

Mohammed Khawaja ◽

Pat Keep ◽

...

Keyword(s):

Capillary Electrophoresis ◽

Prion Protein ◽

Clinical Signs ◽

Test System ◽

Buffy Coat ◽

Infected Sheep ◽

Test Method ◽

Peptide Sequence ◽

Transmissible Spongiform Encephalopathies ◽

Blood Samples

Abstract An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 712 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.

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Seroprevalence of Hepatitis E Virus Infection among Blood Donors in Bulgaria

Viruses ◽

10.3390/v13030492 ◽

2021 ◽

Vol 13 (3) ◽

pp. 492

Author(s):

Magdalena Baymakova ◽

Krasimira Terzieva ◽

Rumen Popov ◽

Elisaveta Grancharova ◽

Todor Kundurzhiev ◽

...

Keyword(s):

Virus Infection ◽

Hepatitis E Virus ◽

Blood Donors ◽

Hepatitis E ◽

Elisa Test ◽

Blood Samples ◽

Wild Boars ◽

Domestic Pigs ◽

Specific Questionnaire ◽

Positive Results

Hepatitis E virus (HEV) infection is widespread among domestic pigs, industrial swine, and wild boars in Bulgaria. The aim of the current research was to present the HEV seroprevalence among blood donors in Bulgaria. In the present study, 555 blood donors (479 males and 76 females) were enrolled from five districts in the country (Shumen, Pleven, Stara Zagora, Plovdiv, and Sofia districts). All blood samples were tested for anti-HEV IgG using the recomWell HEV IgG ELISA test (Mikrogen GmbH, Neuried, Germany). Each participating donor completed a short, structured, and specific questionnaire to document data on the current study. Anti-HEV IgG positive results were detected in 144 (25.9%) blood donors, including 129 (26.9%) males and 15 (19.7%) females. The established HEV seropositivity was 28.8% (23/80) in Shumen district, 23.2% (22/95) in Pleven district, 27.1% (38/140) in Stara Zagora district, 27.5% (44/160) in Plovdiv district, and 21.3% (17/80) in Sofia district. A high HEV seroprevalence was found for persons who declared that they were general hunters (48.7%; 19/39; p = 0.001) and hunters of wild boars (51.6%; 16/31; p = 0.001). We present the first seroprevalence rates of HEV infection in blood donors from Bulgaria. The results of our research showed high HEV seropositivity among blood donors.

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COMPARISON OF FOURTH GENERATION ELISA AND RAPID DIAGNOSTIC TEST FOR DIAGNOSIS OF HEPATITIS C VIRUS(HCV) INFECTION IN A TERTIARY CARE HOSPITAL.

10.36106/2000717 ◽

2021 ◽

pp. 15-17

Author(s):

Stuti Kansra Arora ◽

Mala Chhabra ◽

Anuradha Anuradha ◽

Arvind Achra ◽

Nandini Duggal

Keyword(s):

Diagnostic Test ◽

Rapid Diagnostic Test ◽

Sensitivity And Specificity ◽

Treatment Initiation ◽

Tertiary Care ◽

Hcv Infection ◽

Fourth Generation ◽

World Population ◽

Test Results ◽

Blood Samples

Introduction:Hepatitis C virus (HCV) infection has emerged as one of the major global health challenge affecting about 2 - 3% of the world population. Epidemiological studies have shown that HCV infection is a major risk factor for development of Acute hepatitis,chronic liver disease,cirrhosis and Hepatocellular carcinoma (HCC).Early diagnosis of HCV is important to link hepatitis testing to care and treatment initiation. Aim:To compare sensitivity and specificity of rapid diagnostic test (RDT) with fourth generation ELISA Material and Method: This study was conducted in the Department of Microbiology at Atal Bihari Vajpayee Institute of Medical Sciences (formerly Post Graduate Institute of Medical Education and Research) and Dr Ram Manohar Lohia Hospital from January 2018 to December 2018.Blood samples of patients suspected with hepatitis were tested using ELISA and rapid diagnostic test Results: In our study 26378 blood samples were tested for HCV,using fourth generation ELISA.Of these,581(2.20%) samples were found to be positive by ELISA.These HCV positive samples along with equal number of ELISA negative samples were tested by rapid diagnostic test. Sensitivity and specificity of the rapid diagnostic test was found to be 72.98 % and 100% respectively. Discussion:Rapid diagnostic test can be used during emergency hours but their results must be followed by ELISA test results in a tertiary care hospital.Reporting of false negative results should be minimized for rapid linkage to treatment initiation and to avoid silent transmission of infection.

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The influence of sepsis as a complication after trauma on immune response to injury

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh1104179s ◽

2011 ◽

Vol 139 (3-4) ◽

pp. 179-184

Author(s):

Maja Surbatovic ◽

Darko Mirkovic ◽

Sonja Radakovic ◽

Miodrag Jevtic ◽

Nikola Filipovic

Keyword(s):

Immune Response ◽

Mortality Rate ◽

Inflammatory Response ◽

Proinflammatory Cytokine ◽

Inflammatory Cytokine ◽

Elisa Test ◽

Sepsis Group ◽

Blood Samples ◽

Significant Difference ◽

Anti Inflammatory Cytokine

Introduction. Mortality rate in trauma complicated with sepsis is exceeding 50%. Outcome is not determined only by infection or trauma, but also by the intensity of immuno-inflammatory response. Objective. The aim of this study was to determine the influence of sepsis on the immuno-inflammatory response, in the group of 35 traumatized men, of which in 25 cases trauma was complicated with sepsis. Methods. Cytokines were measured by ELISA test in plasma. Blood samples were drown on the first, third and fifth day after ICU admission. Results. Proinflammatory cytokine IL-8 was 230-fold higher in trauma + sepsis group (1148.48 vs. 5.05 pg/ml; p<0.01), and anti- inflammatory cytokine IL-1ra was 4-fold higher (1138.3 vs. 310.05 pg/ml; p<0.01), whereas IL-12 and IL-4 showed no significant difference between the groups. Conclusion. We concluded that sepsis, as a complication after trauma, drastically enhances immuno-inflammatory response to insult, as indicated by IL-8 and IL-1ra, but not IL-12 and IL-4.

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Identifikasi Keragaman Gen FTO Pada Bangsa Sapi Potong Indonesia

Jurnal Ilmu dan Teknologi Peternakan Tropis ◽

10.33772/jitro.v6i2.5655 ◽

2019 ◽

Vol 6 (2) ◽

pp. 232

Author(s):

Sutikno Sutikno ◽

Rudy Priyanto ◽

Cece Sumantri ◽

Jakaria Jakaria

Keyword(s):

Beef Cattle ◽

Genetic Markers ◽

Direct Sequencing ◽

Nucleotide Position ◽

Fto Gene ◽

Blood Samples ◽

Pcr Rflp ◽

Gene Functions ◽

Hardy Weinberg Equilibrium ◽

Cattle Blood

ABSTRAK Gen FTO berfungsi sebagai regulasi homeostasis, deposisi lemak dan pengaturan obesitas. Penelitian ini bertujuan untuk mengidentifikasi polimorfisme SNP g.125550A>T di ekson 3 gen FTO pada bangsa sapi potong Indonesia. Sampel darah diperoleh dari 209 ekor sapi, terdiri atas sapi bali (44), madura (20), Pesisir (20), katingan (20), Peranakan ongole (PO) (22), Pasundan (20), Sumba Ongole (SO) (11), brahman (20), simental (15), dan limousin (18). Polimorfisme gen FTO dianalisis menggunakan metode PCR-RFLP (HpyCH4III) dan direct sequencing. Hasil genotiping SNP g.125550A>T adalah polimorfik (genotipe AA, AT, dan TT) pada sapi madura, pesisir, katingan, PO, pasundan, SO, brahman, simental, dan limousin. Frekuensi alel A dan T masing-masing adalah 0,70, 0,68, 0,84, 0,89, 0,70, 0,86, 0,90, 0,73, 0,69 dan 0,30, 0,33, 0,16, 0,11, 0,30, 0,14, 0,10, 0,27, 0,31. Nilai Ho dan He masing-masing adalah 0,60-0,14 dan 0,44-0,18 serta dalam keseimbangan Hardy-Weinberg (P>0.05). Sementara pada sapi bali bersifat monomorfik hanya bergenotipe AA. Hasil sekuensing SNP g.125550A>T ditemukan mutasi tranvesi A menjadi T pada posisi nukleotida g.125550. Berdasarkan hasil penelitian ini, dapat disimpulkan bahwa SNP 125550A>T gen FTO beragam dan berpotensi dijadikan marka genetik untuk kualitas daging pada bangsa sapi potong Indonesia.Kata Kunci: gen FTO, PCR-RFLP, Sapi, SNP g.125550A>TABSTRACTThe FTO gene functions as regulation of homeostasis, fat deposition and regulation of obesity. This study aimed to identify the polymorphism of SNP g.125550A>T in exon 3 of FTO gene in Indonesian beef cattle. Blood samples were collected from 209 cattle, including bali (44), madura (20), pesisir (20), katingan (20), PO (22), pasundan (20), SO (11), brahman (20), simental (15), and limousin (18). Polymorphism of the FTO gene was analyzed using PCR-RFLP (HpyCH4III) and direct sequencing methods. The results of genotyping SNP g.125550A>T was polymorphic (AA, AT and TT genotypes) in madura, pesisir, katingan, PO, pasundan, SO, brahman, simental, and limousin cattle. The frequency of A and T alleles were 0,70, 0,68, 0,84, 0,89, 0,70, 0,86, 0,90, 0,73, 0,69 and 0,30, 0,33, 0,16, 0,11, 0,30, 0,14, 0,10, 0,27, 0,31 respectively. The values of Ho and He were 0,60-0,14 and 0,44-0,18 respectively and in Hardy-Weinberg equilibrium (P>0,05). While in Bali cattle was monomorphic (AA genotype). Results of sequencing SNP g.125550A>T of the FTO gene found a transverse mutation A to T at the nucleotide position g.125550. As a result of this study, it can be concluded that SNP 125550A>T of the FTO gene was diverse and potentially used as genetic markers for meat quality in Indonesian beef cattle.Keywords: cattle, FTO gene, PCR-RFLP, SNP g.125550A>T.

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