scholarly journals Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors

2010 ◽  
Vol 43 (2) ◽  
pp. 111-115 ◽  
Author(s):  
Rafaela Gomes Andrade ◽  
Maísa Aparecida Ribeiro ◽  
Maria Sueli Silva Namen-Lopes ◽  
Sônia Mara Nunes Silva ◽  
Fernando Valadares Basques ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Blood Donors ◽  
High Rate ◽  
Confirmatory Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human T Cell ◽  
Blood Banks ◽  
Pcr Assays

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

scholarly journals Contradictory results of serological confirmatory test and real-time PCR assays in diagnosis a patient of HIV-1 infection

2018 ◽  
Vol 74 ◽  
pp. 38-40
Author(s):  
Li Shen ◽  
Xiaoxia Liu ◽  
Ting Wang ◽  
Jing Yang ◽  
Xiao Wu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Confirmatory Test ◽  
Pcr Assays ◽  
Hiv 1

scholarly journals HTLV screening using chemiluminescence immunoassay among blood donors in three blood centers in China

2020 ◽  
Author(s):  
Junpeng Zhao ◽  
Feixue Zhao ◽  
Wei Han ◽  
Xiaoxuan Xu ◽  
Lilin Wang ◽  
...  
Keyword(s):  
Blood Donors ◽  
Confirmatory Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Screening Methods ◽  
Chemiluminescence Immunoassay ◽  
Blood Donations ◽  
High School Degree ◽  
Blood Center ◽  
Human T Cell ◽  
Blood Screening

Abstract Background Human T-cell lymphotropic virus (HTLV) remains a concern for blood safety. The detection of HTLV has not yet been applied for routine blood screening in China, even though lots of HTLV positive cases have been reported in southeastern China. However, data on the prevalence of HTLV infection among blood donors is limited. Objective The objective was to investigate the prevalence of HTLV among blood donors in three representative blood centers in China and evaluate the feasibility of chemiluminescence immunoassay (CLIA) for blood screening. Methods From November 2018 to March 2019, blood plasma samples collected from Hebei, Changsha and Shenzhen blood centers were screening for HTLV-1/2 antibody using Lumipulse G HTLV-I/II Immunoreaction Cartridges set (CLIA) and enzyme-linked immunosorbent assay (ELISA), followed with confirmatory test using INNO-LIA HTLV I/II. Results A total of 59929 blood donations were collected and screened for HTLV-1/2. The reactive rate of CLIA and ELISA among donations in Shenzhen blood center (0.0943%, 27/28621) was higher than Hebei (0.0248%, 4/16144) and Changsha (0.0198%, 3/15164) ( P <0.05). After confirmation, 3 samples were confirmed as indeterminate for HTLV antibodies, only 1 sample from Shenzhen blood center was confirmed and typed as HTLV-1. The overall prevalence of HTLV-1/2 was 1.67 per 100,000 (1/59929). The HTLV-infected blood donor was a first female donor at age 32 with high school degree, born in Fujian province, and SHE minority. Conclusion In summary, the overall prevalence of HTLV-1/2 among blood donors in the tree blood centers in China remains relatively low. However, blood donations with positive or indeterminate results for HTLV antibodies found in the study reminded us the importance of HTLV screening among blood donors in China. In the view of HTLV prevalence and cost, all the first-blood donors should undergo HTLV screening as a systematic strategy to reduce the risk of transmission of HTLV through blood transfusion.

scholarly journals Seroprevalencia de HTLV1/2 en donantes de sangre, Boyacá - Colombia, 2011-2013

2016 ◽  
Vol 18 (2) ◽  
pp. 209
Author(s):  
Mabel Idaliana Medina-Alfonso ◽  
Sandra Marina Forero-Pulido ◽  
Román Yesid Ramírez-Rueda
Keyword(s):  
T Cell ◽  
Western Blot ◽  
Blood Donors ◽  
Cell Lymphoma ◽  
National Level ◽  
Spastic Paraparesis ◽  
Human T Cell ◽  
Blood Donor Screening ◽  
Blood Banks ◽  
Means Of Transmission

Introducción: El virus linfotrópico humano de células T es un retrovirus, se encuentra asociado al riesgo de presentar dermatitis infecciosa, uveítis, polimiositis, artropatías, paraparesia espástica tropical, linfoma de células T del adulto y mieloma. Una de las vías de transmisión del HTLV 1/2 es la transfusión sanguínea de unidades que contienen el virus. Para evitar la transmisión de la infección por este retrovirus, el tamizaje en muestras de donantes de sangre ha sido obligatorio en muchos países y más recientemente en Colombia. Objetivo: Reportar la seroprevalencia de virus HTLV 1/ 2 en donantes de sangre de Boyacá-Colombia entre los años 2011 a 2013. Materiales y Métodos: Estudio descriptivo retrospectivo, en el que se estableció la seroprevalencia de HTLV 1/2 en 48.782 donantes de sangre captados en el departamento de Boyacá por tres bancos de sangre, utilizando la técnica quimioluminiscencia y como pruebas confirmatorias la técnica de InmunoBlot. Resultados: De los donantes tamizados, el 0,23% (113/48.782) fueron serorreactivos (positivos en la prueba de quimioluminiscencia), el 0,16% (78/48.782) fueron negativos, un 0,04% (21/48.782) fueron indeterminados y el 0,03% (14/48.782) fueron seropositivos por la técnica de InmunoBlot. Los donantes seropositivos correspondieron según el sexo a 0,016% (8/48.782) mujeres y 0,012% (6/48.782) a hombres. Conclusión: La prevalencia de HTLV 1/2 fue de 0,03%, similar a la de la mayoría de países que cuentan con reporte de esta determinación en donantes de sangre. Los datos generados en el estudio son los primeros reportados para Boyacá y aportan a la epidemiología del virus a nivel nacional. AbstractIntroduction: The Human T-cell lymphotropic virus is a retrovirus found to be associated with risks of infectious dermatitis, uveitis, polymyositis, arthropathies, tropical spastic paraparesis, myeloma and T-cell lymphoma. One of the means of transmission of HTLV 1/2 is the transfusion of blood units contaminated by the virus. To prevent this kind of transmission, blood donor screening has recently become a standard in Colombia and in many other countries. Objective: To report the seroprevalence of HTLV 1/2 virus in blood donors from Boyacá between 2011 and 2013. Materials and Methods: A retrospective descriptive study was performed, in which the seroprevalence of HTLV 1/2 was established in 48,782 blood donors from three blood banks in the department of Boyacá by using both chemiluminescence and western blot techniques. Results: From the screened donors, 0.23% (113 / 48,782) were seroreactive (chemiluminescence positive), 0.16% (78 / 48,782) were negative, 0.04% (21 / 48,782) were undefined and 0.03% (14 / 48,782) were seropositive by western blot. Concerning gender, seropositive donors were 0.016% (8/48,782) female and 0.012% (6 / 48,782) male. Conclusion: The prevalence of HTLV 1/2 was 0.03% which is similar to what has been found in most of the countries. The data generated in this study is the first of this kind available for Boyacá and will likely contribute to the virus epidemiology at the national level.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 656
Author(s):  
Konstantin Tanida ◽  
Andreas Hahn ◽  
Kirsten Alexandra Eberhardt ◽  
Egbert Tannich ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Real Time ◽  
Indigenous People ◽  
Real Time Pcr ◽  
Latent Class ◽  
Enterocytozoon Bieneusi ◽  
Enteric Disease ◽  
Immunosuppressed Patients ◽  
Study Population ◽  
Stool Samples ◽  
Pcr Assays

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

scholarly journals Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ellen Kathrin Link ◽  
Matthias Eddicks ◽  
Liangliang Nan ◽  
Mathias Ritzmann ◽  
Gerd Sutter ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Diagnostic Sensitivity ◽  
Locked Nucleic Acid ◽  
Porcine Circovirus ◽  
Amplification Efficiency ◽  
Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

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