Effective Management of Hepatitis C Molecular Testing Improves Test Use Without Compromising Patient Management

2002 ◽  
Vol 126 (1) ◽  
pp. 100-102
Author(s):  
Sara Kukuczka ◽  
Leonard E. Grosso
Keyword(s):  
Hepatitis C ◽  
Patient Management ◽  
Cost Effective ◽  
Turnaround Time ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Testing Protocol ◽  
Viral Particles ◽  
Clinically Significant ◽  
Test Use

Abstract Context.—The availability of effective antiviral therapy for hepatitis C has increased the need for molecular detection and quantification of circulating hepatitis C viral particles. The limits of detection differ for the quantitative and qualitative reverse transcriptase polymerase chain reaction (RT-PCR) assays; furthermore, adequate patient assessment requires both detection of hepatitis C virus when it is present and quantitation of the viral load when possible. The combination of these factors promotes the simultaneous ordering of both tests with the possibility of generating redundant test information. Objective.—To reduce the number of unnecessary hepatitis C tests performed. Methods.—We established a reflexive testing protocol for quantitative and qualitative RT-PCR testing for hepatitis C. Results.—During a 3½-month interval, 170 qualitative RT-PCR hepatitis C tests were eliminated (a 59.4% reduction in the number of these tests). This reduction was achieved without a clinically significant change in turnaround time or a compromise of patient care. Conclusions.—Establishing the quantitative and qualitative RT-PCR tests in-house and adopting the reflexive testing protocol was cost-effective and did not compromise patient management or care.

scholarly journals ECNano: A Cost-Effective Workflow for Target Enrichment Sequencing and Accurate Variant Calling on 4,800 Clinically Significant Genes Using a Single MinION Flowcell

2021 ◽  
Author(s):  
Amy Wing-Sze Leung ◽  
Henry Chi-Ming Leung ◽  
Chak-Lim Wong ◽  
Zhen-Xian Zheng ◽  
Wui-Wang Lui ◽  
...  
Keyword(s):  
Variant Calling ◽  
Cost Effective ◽  
Turnaround Time ◽  
Read Length ◽  
Sequencing Error ◽  
Target Enrichment ◽  
Long Read ◽  
Wet Lab ◽  
Variant Detection ◽  
Clinically Significant

Background: The application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample. Method: We introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4,800 clinically important genes and regions using a single MinION flowcell. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples. Results: ECNano achieved deep on-target depth of coverage (DoC) at average >100x and >98% uniformity using one MinION flowcell. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30x DoC. ECNano obtained an average read length of 1,000 bp. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having ≥ 30x DoC. Clair-ensemble achieved >99% recall and accuracy for SNV calling. The whole workflow from wet-lab protocol to variant detection was completed within three days. Conclusion: We presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4,800 clinically significant genes and regions using a single MinION flowcell. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction.

scholarly journals A scalable saliva-based, extraction-free rt-lamp protocol for sars-cov-2 diagnosis

2020 ◽  
Author(s):  
Paula Asprino ◽  
Fabiana Bettoni ◽  
Anamaria Camargo ◽  
Diego Coelho ◽  
Guilherme Coppini ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Cost Effective ◽  
Turnaround Time ◽  
Curve Analysis ◽  
Nasopharyngeal Swab ◽  
Screening Methods ◽  
Rt Pcr ◽  
Effective Screening ◽  
Point Curve ◽  
Average Turnaround Time

I.ABSTRACTScalable, cost-effective screening methods are an essential tool to control SARS-CoV-2 spread. We have developed a straight saliva-based, RNA extraction-free, RT-LAMP test that is comparable to current nasopharyngeal swab RT-PCR tests in both sensitivity and specificity. Using a 2-step readout of fluorescence and melting-point curve analysis, the test is scalable to more than 30,000 tests per day with average turnaround time of less than 3 hours. The test was validated using samples from 244 symptomatic patients, and showed sensitivity of 78.9% (vs. 85.5% for nasopharyngeal swabs RT-PCR) and specificity of 100% (vs. 100% for nasopharyngeal swabs RT-PCR). Our method is therefore accurate, robust, time and cost effective and therefore can be used for screening of SARS-CoV-2.

scholarly journals Hepatitis C Virus Quantitation: Optimization of Strategies for Detecting Low-Level Viremia

2000 ◽  
Vol 38 (2) ◽  
pp. 888-891 ◽  
Author(s):  
Wolfgang T. Hofgärtner ◽  
Jeffrey A. Kant ◽  
Karen E. Weck
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Medical Center ◽  
Significant Proportion ◽  
Cost Effective ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Economic Implications ◽  
Hcv Testing

A long-term assessment of quantitative hepatitis C virus (HCV) testing was performed at the University of Pittsburgh Medical Center. The Quantiplex HCV RNA 2.0 branched-chain DNA (bDNA) assay (Bayer Diagnostics) for hepatitis C viral load determination was used to test 3,471 specimens. bDNA-negative samples were also tested by an in-house qualitative reverse transcriptase (RT)-PCR assay with a measured sensitivity of fewer than 100 HCV genome equivalents per milliliter. Of 1,239 bDNA-negative specimens, 74.1% were negative and 25.9% were positive by RT-PCR, indicating the presence of viremia in a significant proportion of bDNA-negative samples. We discuss the medical and economic implications of these results and propose two alternatives for clinical laboratories to consider in approaching quantitative HCV testing. For laboratories able to perform a sensitive RT-PCR assay for ≤40% of the bDNA test cost, prescreening bDNA requests by RT-PCR may be the most cost-effective approach.

scholarly journals Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fernanda de Mello Malta ◽  
Deyvid Amgarten ◽  
Felipe Camilo Val ◽  
Murilo Castro Cervato ◽  
Bruna Mascaro Cordeiro de Azevedo ◽  
...  
Keyword(s):  
Gold Standard ◽  
Rna Extraction ◽  
Cost Effective ◽  
Single Step ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Major Bottleneck ◽  
Wet Lab ◽  
Coronavirus Infection ◽  
Performance Results

AbstractSince the first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome was deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription—Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies, such as Brazil. Additionally, RT-qPCR scalability is highly dependent on equipment that usually performs testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-qPCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient’s samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-qPCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6144 samples in a single run. Performance results when compared with RT-qPCR show general accuracy of 96%, and 98% when only samples with CT values (gene N) lower than 30 are considered. We have also developed an online platform, termed VarsVID, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets generation, sample sheet for sequencing and results’ display are all features provided by VarsVID. Altogether, these results will contribute to control COVID19 pandemics.

scholarly journals Predictive molecular pathology in the time of coronavirus disease (COVID-19) in Europe

2020 ◽  
pp. jclinpath-2020-206957 ◽  
Author(s):  
Umberto Malapelle ◽  
Pasquale Pisapia ◽  
Antonino Iaccarino ◽  
Massimo Barberis ◽  
Claudio Bellevicine ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Liquid Biopsy ◽  
Predictive Biomarker ◽  
Turnaround Time ◽  
Molecular Testing ◽  
Advanced Stage ◽  
Molecular Test ◽  
Rt Pcr ◽  
Molecular Tests ◽  
Advanced Stage Lung Cancer

AimsLung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak.MethodsFifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time.ResultsIn most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing.ConclusionsThe workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

scholarly journals Saliva-Dry LAMP: A Rapid Near-Patient Detection System for SARS-CoV-2.

2021 ◽  
Author(s):  
Noah Toppings ◽  
Abu Mohon ◽  
Yoonjung Lee ◽  
Hitendra Kumar ◽  
Daniel Lee ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Molecular Testing ◽  
Fluorometric Detection ◽  
Analytical Sensitivity ◽  
Sample Collection ◽  
Rt Pcr ◽  
Percent Agreement

Abstract The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. The study sought to develop a rapid, near-patient saliva-based test for COVID-19 with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved positive percent agreement of 100% [95% CI 88.43% to 100.0%] and negative percent agreement of 96.7% [95% CI 82.78% to 99.92%] on saliva. Saliva-Dry LAMP can be completed in 105 minutes. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

scholarly journals A rapid near-patient detection system for SARS-CoV-2 using saliva

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Noah B. Toppings ◽  
Abu Naser Mohon ◽  
Yoonjung Lee ◽  
Hitendra Kumar ◽  
Daniel Lee ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Detection System ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Molecular Testing ◽  
Fluorometric Detection ◽  
Analytical Sensitivity ◽  
Sample Collection ◽  
Rt Pcr ◽  
Percent Agreement

AbstractThe highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78–99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.

scholarly journals Noninvasive Assessment of Hepatitis C Virus Infected Patients Using Vibration-Controlled Transient Elastography

2021 ◽  
Vol 10 (12) ◽  
pp. 2575
Author(s):  
Mira Florea ◽  
Teodora Serban ◽  
George Razvan Tirpe ◽  
Alexandru Tirpe ◽  
Monica Lupsor-Platon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Transient Elastography ◽  
Ultrasonic Attenuation ◽  
Antiviral Treatment ◽  
Liver Stiffness ◽  
Cost Effective ◽  
Ultrasound Elastography ◽  
Accurate Identification ◽  
Clinically Significant

Chronic infection with hepatitis C virus (HCV) is one of the leading causes of cirrhosis and hepatocellular carcinoma (HCC). Surveillance of these patients is an essential strategy in the prevention chain, including in the pre/post-antiviral treatment states. Ultrasound elastography techniques are emerging as key methods in the assessment of liver diseases, with a number of advantages such as their rapid, noninvasive, and cost-effective characters. The present paper critically reviews the performance of vibration-controlled transient elastography (VCTE) in the assessment of HCV patients. VCTE measures liver stiffness (LS) and the ultrasonic attenuation through the embedded controlled attenuation parameter (CAP), providing the clinician with a tool for assessing fibrosis, cirrhosis, and steatosis in a noninvasive manner. Moreover, standardized LS values enable proper staging of the underlying fibrosis, leading to an accurate identification of a subset of HCV patients that present a high risk for complications. In addition, VCTE is a valuable technique in evaluating liver fibrosis prior to HCV therapy. However, its applicability in monitoring fibrosis regression after HCV eradication is currently limited and further studies should focus on extending the boundaries of VCTE in this context. From a different perspective, VCTE may be effective in identifying clinically significant portal hypertension (CSPH). An emerging prospect of clinical significance that warrants further study is the identification of esophageal varices. Our opinion is that the advantages of VCTE currently outweigh those of other surveillance methods.

scholarly journals Mass molecular testing for COVID19 using NGS-based technology and a highly scalable workflow

2020 ◽  
Author(s):  
Fernanda de Mello Malta ◽  
Deyvid Emanuel Amgarten ◽  
Felipe Camilo Val ◽  
Rubia Anita Ferraz Santana ◽  
Murilo Castro Cervato ◽  
...  
Keyword(s):  
Gold Standard ◽  
Rna Extraction ◽  
Cost Effective ◽  
Single Step ◽  
Molecular Testing ◽  
Rt Pcr ◽  
Major Bottleneck ◽  
Wet Lab ◽  
Coronavirus Infection ◽  
Performance Results

Since first reported case of the new coronavirus infection in Wuhan, China, researchers and governments have witnessed an unseen rise in the number of cases. Thanks to the rapid work of Chinese scientists, the pathogen now called SARS-CoV-2 has been identified and its whole genome has been deposited in public databases by early January 2020. The availability of the genome has allowed researchers to develop Reverse Transcription - Polymerase Chain Reaction (RT-PCR) assays, which are now the gold-standard for molecular diagnosis of the respiratory syndrome COVID19. Because of the rising number of cases and rapid spreading, the world has been facing a shortage of RT-PCR supplies, especially the ones involved in RNA extraction. This has been a major bottleneck to increase testing capacity in many countries that do not significantly manufacture these supplies (Brazil included). Additionally, RT-PCR scalability is highly dependent on equipment that usually perform testing of 96 samples at a time. In this work, we describe a cost-effective molecular NGS-based test for diagnosis of COVID19, which uses a single-step RNA extraction and presents high scalability and accuracy when compared to the gold-standard RT-PCR. A single run of the NGS-based test using the Illumina NextSeq 550 mid-end sequencing equipment is able to multiplex 1,536 patient samples, providing individual semi-qualitative results (detected, not detected). Detected results are provided with fragments per million (FPM) values, which was demonstrated to correlate with RT-PCR Cycle Threshold (CT) values. Besides, usage of the high-end Illumina Novaseq platform may yield diagnostic for up to 6,144 samples in a single run. Performance results when compared with RT-PCR show general accuracy of 96% (or 98% when only samples with CT values for gene N lower than 30 are considered). We have also developed an online platform, called VarsVID, a Varstation feature, to help test executors to easily scale testing numbers. Sample registering, wet-lab worksheets, sample sheet for sequencing and results display are all features provided by VarsVID on Varstation. Altogether, these results will contribute to control COVID19 pandemics.

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