scholarly journals Computer-Aided Medical Microbiology Monitoring Tool: A Strategy to Adapt to the SARS-CoV-2 Epidemic and That Highlights RT-PCR Consistency

2021 ◽  
Vol 11 ◽  
Author(s):  
Linda Mueller ◽  
Valentin Scherz ◽  
Gilbert Greub ◽  
Katia Jaton ◽  
Onya Opota
Keyword(s):  
Turnaround Time ◽  
Quality Monitoring ◽  
Rt Pcr ◽  
Monitoring Tool ◽  
Diagnostic Laboratory ◽  
Important Health ◽  
Polymerase Chain ◽  
Automated Processes ◽  
Regulatory Decisions ◽  
Case Validation

Since the beginning of the COVID-19 pandemic, important health and regulatory decisions relied on SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) results. Our diagnostic laboratory faced a rapid increase in the number of SARS-CoV-2 RT-PCR. To maintain a rapid turnaround time, we moved from a case-by-case validation of RT-PCR results to an automated validation and immediate results transmission to clinicians. A quality-monitoring tool based on a homemade algorithm coded in R was developed, to preserve high quality and to track aberrant results. We present the results of this quality-monitoring tool applied to 35,137 RT-PCR results. Patients tested several times led to 4,939 pairwise comparisons: 88% concordant and 12% discrepant. The algorithm automatically solved 428 out of 573 discrepancies. The most likely explanation for these 573 discrepancies was related for 44.9% of the situations to the clinical evolution of the disease, 27.9% to preanalytical factors, and 25.3% to stochasticity of the assay. Finally, 11 discrepant results could not be explained, including 8 for which clinical data was not available. For patients repeatedly tested on the same day, the second result confirmed a first negative or positive result in 99.2% or 88.9% of cases, respectively. The implemented quality-monitoring strategy allowed to: i) assist the investigation of discrepant results ii) focus the attention of medical microbiologists onto results requiring a specific expertise and iii) maintain an acceptable turnaround time. This work highlights the high RT-PCR consistency for the detection of SARS-CoV-2 and the necessity for automated processes to handle a huge number of microbiological results while preserving quality.

scholarly journals Computer-aided medical microbiology monitoring tool: a strategy to adapt to the SARS-CoV-2 epidemic and that highlights RT-PCR consistency

2020 ◽  
Author(s):  
Linda Mueller ◽  
Valentin Scherz ◽  
Gilbert Greub ◽  
Katia Jaton ◽  
Onya Opota
Keyword(s):  
Turnaround Time ◽  
Quality Monitoring ◽  
Rt Pcr ◽  
Monitoring Tool ◽  
Diagnostic Laboratory ◽  
Health Authorities ◽  
Important Health ◽  
Public Health Authorities ◽  
Automated Processes ◽  
Case Validation

Since the beginning of the COVID-19 pandemic, important health and regulatory decisions relied on the SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) results. Our diagnostic laboratory faced a rapid increase in the number of SARS-CoV-2 RT-PCR, with up to 1,007 tests per day. To maintain a rapid turnaround time to support patient management and public health authorities' decisions, we moved from a case-by-case validation of RT-PCR to an automated validation and immediate transmission of the results to clinicians. To maintain high quality and to track possible aberrant results, we developed a quality-monitoring tool based on a homemade algorithm coded in R. We present the results of this quality-monitoring tool applied to 35,137 RT-PCR results corresponding to 30,198 patients. Patients tested several times led to 4,939 pairwise comparisons; 88% concordant and 12% discrepant. Among the 573 discrepancies, 428 were automatically solved by the algorithm. The most likely explanation for these 573 discrepancies was related for 44.9% of the situations to "Clinical evolution", 27.9% to "Preanalytical" problems, and 25.3% to "Stochastic". Finally, 11 discrepant results could not be explained, including 8 received from external partners for which clinical data were not available. The implemented quality-monitoring strategy allowed to: i) assist the investigation of discrepant results ii) focus the attention of medical microbiologists onto results requiring a specific expertise and iii) maintain an acceptable TAT. This work highlighted the high RT-PCR consistency for the detection of SARS-CoV-2 and the importance of automated processes to handle a huge number of samples while preserving quality.

scholarly journals Detection of Bovine Torovirus and other Enteric Pathogens in Feces from Diarrhea Cases in Cattle

2003 ◽  
Vol 15 (3) ◽  
pp. 205-212 ◽  
Author(s):  
Armando E. Hoet ◽  
Paul R. Nielsen ◽  
Mustafa Hasoksuz ◽  
Christopher Thomas ◽  
Thomas E. Wittum ◽  
...  
Keyword(s):  
Etiologic Agent ◽  
Enteric Pathogens ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Salmonella Spp ◽  
Diagnostic Laboratory ◽  
Fecal Samples ◽  
Polymerase Chain

The objectives of this study were to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples from diarrhea cases submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) and to assess if a relationship exists between BoTV and the other enteric pathogens detected. From November 1999 to May 2001, 259 specimens from 53 calves (≤6 months old), 27 young adults (≤2 years), 125 adults (≥2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect BoTV Testing for other enteric pathogens was performed by ADDL, and the results were analyzed with the BoTV data. The BoTV was detected using ELISA or RT-PCR in 9.7% (25/259) of the clinical samples, 56% (14/25) of which were from calves ( P < 0.001) representing 26.4% (14/53) of the calves tested. Of the BoTV-positive calves, 71% (10/14) were less than 3 weeks of age. In 11/25 positive specimens, BoTV was the only pathogen detected among those examined. Other enteric organisms detected alone or in combination with BoTV in calf samples were rotavirus, coronavirus, Salmonella spp., Cryptosporidium spp., and Giardia spp.; but no consistent association between BoTV and these organisms was observed. In summary, BoTV was detected in fecal samples from cattle with diarrhea, principally in young calves less than 3 weeks of age. Future studies of infectious diarrhea in cattle should also include assays for this etiologic agent.

Removal of norovirus from water by coagulation, flocculation and sedimentation processes

2014 ◽  
Vol 15 (1) ◽  
pp. 158-163 ◽  
Author(s):  
Gwy-Am Shin ◽  
Mark D. Sobsey
Keyword(s):  
Norwalk Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Treatment Processes ◽  
Physico Chemical ◽  
Important Health ◽  
Health Related ◽  
Polymerase Chain ◽  
The One ◽  
Sedimentation Processes

In this study, we determined the removal of a prototype human norovirus (Norwalk virus, NV) by bench-scale alum coagulation, flocculation and sedimentation processes using reverse transcriptase polymerase chain reaction (RT-PCR) for norovirus assays. After determining optimum conditions for the coagulation, flocculation and sedimentation processes in terms of turbidity reduction, jar tests were performed using the same waters seeded with test viruses. For comparison, two other important health-related viruses, poliovirus 1 (PV1) and coliphage MS2, were included in this study. The removal of NV by coagulation, flocculation and sedimentation processes based on RT-PCR assay in this study was 1.5 log10, which was similar to that of PV1 and a little lower than that of coliphage MS2 (2 log10) based on the same RT-PCR assay. The removal of NV in this study (1.5 log10) is considerably higher than the one in a recent study using recombinant norovirus virus-like particles (∼0.7 log10). Overall, the results of this study suggest that human noroviruses can be appreciably reduced by a properly-operated coagulation, flocculation and sedimentation processes and the contamination of drinking water by noroviruses should be controlled by conventional water treatment processes with conventional physico-chemical processes and disinfection.

scholarly journals Detection of Avian Influenza Virus of H9 Subtype in the Faeces of Experimentally and Naturally Infected Chickens by Reverse Transcription-Polymerase Chain Reaction

2007 ◽  
Vol 76 (3) ◽  
pp. 405-413 ◽  
Author(s):  
H. Noroozian ◽  
M. Vasfi Marandi ◽  
M. Razazian
Keyword(s):  
Polymerase Chain Reaction ◽  
Avian Influenza ◽  
Reverse Transcription ◽  
Predictive Value ◽  
Relative Sensitivity ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Diagnostic Laboratory ◽  
Polymerase Chain ◽  
Virus Subtype

Avian Influenza (AI) is a viral, highly contagious disease of domestic and wild birds. In an avian diagnostic laboratory, it is essential to have methods for rapid detection of respiratory viruses. In the present study, cloacal swabs collected from chickens experimentally and naturally infected with mild pathogenicity AI virus subtype H9, used in a reverse transcription-polymerase chain reaction (RTPCR) assay for detection of AI. On cloacal swabs collected from experimentally infected chickens, AI virus was detected most frequently between days 3 and 7 post infection (p.i.) and the relative sensitivity, specificity, correlation rate, positive predictive value and negative predictive value of the RT-PCR compared to virus isolation (VI) assay were 84%, 80%, 82%, 83% and 81%, respectively. On pooled cloacal swabs collected from flocks suspected of AI, these results were 96%, 100%, 97%, 83% and 100%, respectively. The results proved that the RT-PCR assay could be a reliable and rapid alternative to VI assay for detection of AI virus subtype H9 in faecal specimens.

scholarly journals Impact of Enterovirus Molecular Assay Turnaround Time on Hospitalization Length During an Echovirus 30 Meningitis Outbreak, France, Fall 2014

2019 ◽  
Vol 13 (1) ◽  
pp. 1-8
Author(s):  
Yohan N’Guyen ◽  
Anne L. Lebreil ◽  
Philippine Simphal ◽  
Christine Pietrement ◽  
Nathalie Bednarek ◽  
...  
Keyword(s):  
Patient Discharge ◽  
Turnaround Time ◽  
Sequencing Data ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Echovirus 30 ◽  
Simple Regression Analysis ◽  
Polymerase Chain ◽  
Viral Protein 1 ◽  
The Impact

Background: The impact of Enterovirus Real Time-Polymerase Chain Reaction assay (EV RT-PCR) on hospitalization lengths of patients with aseptic meningitis has been investigated but the impact of early EV RT-PCR results released on time before patient discharge remains unclear during Echovirus meningitis outbreaks. Objective: To assess a potential correlation between EV RT-PCR turn-around time and hospitalization lengths during an Echovirus meningitis outbreak. Method: Eighteen patients demonstrating a positive EV RT-PCR assay performed on Cerebrospinal Fluid (CSF) samples collected between October 1st 2014 and December 31st 2014 were retrospectively included. Viral protein 1 (VP1) gene region was amplified and sequenced using a classical Sanger sequencing reaction. Clinical data were retrospectively collected from patient’s records. Quantitative variables expressed as median values and ranges were compared using Mann Whitney U test. Correlations were performed using simple regression analysis. Results: Phylogenetic VP1 sequence analyses identified that the outbreak was related to an Echovirus 30 strain in 7 out of the 10 cases with available sequencing data. The three remaining sequences analyses evidenced Echovirus 14, 9 and 7 strains. Hospitalization length was statistically shorter in children without comorbidity (n=5) than in adult patients (n=10) or neonates and children with comorbidity (n=3) (p=0.003 and 0.01 respectively), whereas EV RT-PCR turnaround time was not statistically different between these groups. Correlation between hospitalization length and EV RT-PCR turnaround time was poor (R2=0.06), especially in adults (R2=0.01) Conclusion: Our data indicated that EV RT-PCR turnaround time was not correlated to hospitalization length during a short Echovirus meningitis outbreak.

scholarly journals Molecular methods for diagnosing novel coronavirus infection: comparison of loop-mediated isothermal amplification and polymerase chain reaction

2022 ◽  
Vol 66 (6) ◽  
pp. 417-424
Author(s):  
V. G. Akimkin ◽  
V. V. Petrov ◽  
K. V. Krasovitov ◽  
N. I. Borisova ◽  
I. A. Kotov ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Turnaround Time ◽  
Diagnostic Methods ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Advantages And Disadvantages ◽  
Coronavirus Infection ◽  
Polymerase Chain ◽  
Novel Coronavirus

Introduction. Currently, the basis for molecular diagnostics of most infections is the use of reverse transcription polymerase chain reaction (RT-PCR). Technologies based on reverse transcription isothermal loop amplification (RT-LAMP) can be used as an alternative to RT-PCR for diagnostic purposes. In this study, we compared the RTLAMP and RT-PCR methods in order to analyze both the advantages and disadvantages of the two approaches.Material and methods. For the study, we used reagent kits based on RT-PCR and RT-LAMP. The biological material obtained by taking swabs from the mucous membrane of the oropharynx and nasopharynx in patients with symptoms of a new coronavirus infection was used.Results. We tested 381 RNA samples of the SARS-CoV-2 virus (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) from various patients. The obtained values of the threshold cycle (Ct) for RT-PCR averaged 20.0 ± 3.7 s (1530 ± 300 s), and for RT-LAMP 12.8 ± 3.7 s (550 ± 160 s). Proceeding from the theoretical assumptions, a linear relationship between values obtained in two kits was proposed as a hypothesis; the correlation coefficient was approximately 0.827. At the same time, for samples with a low viral load (VL), the higher Ct values in RT-LAMP did not always correlated with those obtained in RT-PCR.Discussion. We noted a significant gain in time for analysis using RT-LAMP compared to RT-PCR, which can be important in the context of testing a large number of samples. Being easy to use and boasting short turnaround time, RT-LAMP-based test systems can be used for mass screening in order to identify persons with medium and high VLs who pose the greatest threat of the spread of SARS-CoV-2, while RT-PCR-based diagnostic methods are also suitable for estimation of VL and its dynamics in patients with COVID-19.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

Cloning of the cDNA Encoding Human Platelet CD36: Comparison to PCR Amplified Fragments of Monocyte, Endothelial and HEL Cells

1993 ◽  
Vol 70 (03) ◽  
pp. 500-505 ◽  
Author(s):  
B Wyler ◽  
L Daviet ◽  
H Bortkiewicz ◽  
J-C Bordet ◽  
J L McGregor
Keyword(s):  
Apparent Molecular Weight ◽  
Platelet Glycoprotein ◽  
Rt Pcr ◽  
Post Translational Modifications ◽  
Hel Cells ◽  
Flanking Region ◽  
Polymerase Chain ◽  
A Minor ◽  
Flanking Regions ◽  
Cdna Fragments

SummaryGlycoprotein CD36, also known as GPIIIb or GPIV, is a major platelet glycoprotein that bears the newly identified Naka alloantigen. The aim of this study was to clone platelet CD36 and investigate other forms of CD36-cDNA present in monocytes, endothelial and HEL cells. RNA from above mentioned cells were reverse transcribed (RT), using specific primers for CD36, and amplified by the polymerase chain reaction (PCR) technique. Sequencing the different amplified platelet derived cDNA fragments, spanning the whole coding and flanking regions, showed the near identity between platelet and CD36-placenta cDNA. Platelet CD36-cDNA cross-hybridized, in Southern blots, with RT-PCR amplified cDNA originating from monocytes, endothelial and HEL cells. However, monocytes showed a RT-PCR amplified cDNA fragment (561 bp) that was present in platelets and placenta but not on endothelial on HEL-cells. Northern blot analysis of platelet RNA hybridized with placenta CD36 indicated the presence of a major (1.95 kb) and a minor (0.95 kb) transcript. The 1.95 kb transcript was the only one observed on Northern blots of monocytes, endothelial and HEL cells. These results indicate that the structure of CD36 expressed in platelets is similar, with the exception of the 3’ flanking region, to that of placenta. Differences in apparent molecular weight between CD36 and CD36-like glycoproteins may be due to post-translational modifications.

Expression of Platelet Membrane Glycoprotein V in Human Megakaryocytes and Megakaryocytic Cell Lines: A Study Using a Novel Monoclonal Antibody against GPV

1994 ◽  
Vol 72 (05) ◽  
pp. 762-769 ◽  
Author(s):  
Toshiro Takafuta ◽  
Kingo Fujirmura ◽  
Hironori Kawano ◽  
Masaaki Noda ◽  
Tetsuro Fujimoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Immunohistochemical Analysis ◽  
Specific Protein ◽  
Platelet Membrane ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Flow Cytometric ◽  
Polymerase Chain ◽  
Megakaryocytic Cell

SummaryGlycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myri-state-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.

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