Monitoring of Inhibitors of Enzymatic Amplification in Polymerase Chain Reaction and Evaluation of Efficacy of RNA Extraction for the Detection of Hepatitis C Virus Using the Internal Control

Miyachi, Hayato; Masukawa, Atsuko; Ohshima, Toshio; Fusegawa, Hisae; Hirose, Toru; Impraim, C C; Ando, Yumiko

doi:10.1515/cclm.1998.098

Cited by 14 publications

(7 citation statements)

References 13 publications

(9 reference statements)

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“…Their removal would reduce the number of indeterminate results. Several modi®cations in the nucleic acid extraction procedures might help eliminate these inhibitors [Miyachi et al, 1998;Abu Al-Soud and Radstrom, 2000].…”

Section: Discussionmentioning

confidence: 99%

Screening of blood from potential organ and cornea donors for viruses

Miédougé

Chatelut

Mansuy

et al. 2002

Journal of Medical Virology

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Prospective nucleic acid tests were carried out for human immunodeficiency virus (HIV) and hepatitis C virus (HCV) using the COBAS Amplicor HIV-1 and HCV tests (Roche Diagnostics, Meylan, France) on potential organ (n=113) and cornea (n=368) donors in France to evaluate their performance and suitability for use as a complement to routine serological tests. Blood samples were collected from organ donors with preserved cardiac function after verification of cerebral death. Blood samples were collected from cornea donors post-mortem within 48 hr after death. An internal control was added to the samples before extraction to monitor each individual polymerase chain reaction (PCR). The nucleic acid tests were always interpretable in organ donors and negative in all except in 2 anti-HCV positive patients. One had an indeterminate HIV p24 antigen but was negative for HIV RNA. HIV and HCV RNA were not found in cornea donors with a negative serology but indeterminate molecular results were frequent in this group (17.6%). Cornea donors also gave significantly more (14.4%) indeterminate serological results than organ donors (1.8%) (P<0.001). This was due to the poor quality of the blood samples collected post-mortem. However, there was no correlation between indeterminate results of serological and molecular tests. There were 16/19 (84%) indeterminate serological results for HIV and 4/4 (100%) for HCV that were negative by PCR. Thus, nucleic acid tests could be useful for qualifying a donor whose serological results are indeterminate. The extraction procedures on post-mortem specimens and/or blood collection must be changed to improve the performance of nucleic acid tests.

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Section: Discussionmentioning

confidence: 99%

Screening of blood from potential organ and cornea donors for viruses

Miédougé

Chatelut

Mansuy

et al. 2002

Journal of Medical Virology

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“…Additionally, we considered that anticoagulation might inhibit the PCR for SARS-CoV-2. Some in-vivo studies showed that heparin inhibits different PCRs for other viruses, 24,25 but the evidence of the inhibitory effect of OACs on PCRs for SARS-CoV-2 is lacking. Regardless, in many instances the outcome of a positive COVID-19 test reflects both infection and symptom severity leading to test seeking, the latter of which could have been influenced by anticoagulant activity.…”

Section: Principal Findingsmentioning

confidence: 99%

Association between oral anticoagulants and COVID-19 related outcomes: two cohort studies

Tomlinson

Brown

et al. 2021

Preprint

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ObjectivesWe investigated the role of routinely prescribed oral anticoagulants (OACs) in COVID-19 outcomes, comparing current OAC use versus non-use in Study 1; and warfarin versus direct oral anticoagulants (DOACs) in Study 2.DesignTwo cohort studies, on behalf of NHS England.SettingPrimary care data and pseudonymously-linked SARS-CoV-2 antigen testing data, hospital admissions, and death records from England.ParticipantsStudy 1: 70,464 people with atrial fibrillation (AF) and CHA□DS□-VASc score of 2. Study 2: 372,746 people with non-valvular AF.Main outcome measuresTime to test for SARS-CoV-2, testing positive for SARS-CoV-2, COVID-19 related hospital admission, COVID-19 deaths or non-COVID-19 deaths in Cox regression.ResultsIn Study 1, we included 52,416 current OAC users and 18,048 non-users. We observed no difference in risk of being tested for SARS-CoV-2 associated with current use (adjusted HR, 1.01, 95%CI, 0.96 to 1.05) versus non-use. We observed a lower risk of testing positive for SARS-CoV-2 (adjusted HR, 0.73, 95%CI, 0.60 to 0.90), and COVID-19 deaths (adjusted HR, 0.69, 95%CI, 0.49 to 0.97) associated with current use versus non-use. In Study 2, we included 92,339 warfarin users and 280,407 DOAC users. We observed a lower risk of COVID-19 deaths (adjusted HR, 0.79, 95%CI, 0.76 to 0.83) associated with warfarin versus DOACs. Similar associations were found for all other outcomes.ConclusionsAmong people with AF and a CHA□DS□-VASc score of 2, those receiving OACs had a lower risk of receiving a positive COVID-19 test and severe COVID-19 outcomes than non-users; this might be explained by a causal effect of OACs in preventing severe COVID-19 outcomes or more cautious behaviours leading to reduced infection risk. There was no evidence of a higher risk of severe COVID-19 outcomes associated with warfarin versus DOACs in people with non-valvular AF regardless of CHA□DS□-VASc score.Key pointsWhat is already known on this topicCurrent studies suggest that prophylactic or therapeutic anticoagulant use, particularly low molecular weight heparin, lower the risk of pulmonary embolism and mortality during hospitalisation among patients with COVID-19.Reduced vitamin K status has been reported to be correlated with severity of COVID-19. This could mean that warfarin, as a vitamin K antagonist, is associated with more severe COVID-19 disease than non-vitamin K anticoagulants.What this study addsIn 70,464 people with atrial fibrillation, at the threshold of being treated with an OAC based on risk of stroke, we observed a lower risk of testing positive for SARS-CoV-2 and COVID-19 related deaths associated with routinely prescribed OACs, relative to non-use.This might be explained by OACs preventing severe COVID-19 outcomes, or more cautious behaviours and environmental factors reducing the risk of SARS-CoV-2 infection in those taking OACs.In 372,746 people with non-valvular atrial fibrillation, there was no evidence of a higher risk of severe COVID-19 outcomes associated with warfarin compared with DOACs.

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“…In the present study, the internal control was demonstrated to be useful for monitoring for the presence of inhibitors in a patient under hemodialysis as well as in an in vitro interference study. It could be used to determine which samples yield invalid results and therefore need to be retested (10). Since many kinds of clinical specimens, from body fluids to tissues, are subjected to tests for direct detection of infectious agents, development of automated systems is desirable for nucleic acid extraction procedures that can be ap- a Heparin was added to serum containing 10 3 or 10 5 copies of HCV per ml.…”

Section: Discussionmentioning

confidence: 99%

Automated Specific Capture of Hepatitis C Virus RNA with Probes and Paramagnetic Particle Separation

Miyachi¹,

Masukawa²,

Ohshima³

et al. 2000

J Clin Microbiol

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We developed and evaluated a prototype automated specimen preparation instrument for the specific capture of hepatitis C virus (HCV) RNA with probes and magnetic bead-fluid separation. HCV RNA was isolated from serum by lysis of virus particles with a chaotropic agent, followed by hybridization of the RNA with biotinylated probes and capture of the hybridized RNA with streptavidin-coated paramagnetic particles. After washing of the hybrid-particle complexes to remove nonspecifically bound materials, the particles were resuspended in a specimen diluent and were then ready for amplification and detection with a fully automated PCR system (COBAS AMPLICOR; Roche Diagnostic Systems). The analytical sensitivity in the dilution series was 33 copies per ml or greater. Comparison of the test results with those obtained by a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R; Sanko Junyaku Co., Ltd.) showed 93% (49 of 53 samples) sensitivity and 100% (12 of 12 samples) specificity. There was 94% overall agreement between results. When RNA was extracted by the manual method from serum containing 10 3 or 10 5 copies of HCV per ml in the presence of heparin, there was an inhibitory effect on detection of both HCV RNA and the internal control. In contrast, when RNA was extracted from the serum by the automated method, there was no inhibitory effect. This inhibitory effect of heparin on the manual method was also observed for a series of serum specimens from a hemodialysis patient, but the inhibitory effect was eliminated by the automated specimen preparation method. In summary, a fully automated RNA extraction system for PCR detection of HCV RNA by use of specific capture with probes and magnetic bead-fluid separation was shown to have performance similar to that of the conventional manual method. In addition, it successfully eliminated the inhibitory effect of the heparin in the serum and permitted the detection of HCV RNA in serum samples from a hemodialysis patient. The prototype automated RNA extraction system is suitable as a totally automated system, starting with RNA extraction to detection of HCV, if it was combined with the fully automated COBAS AMPLICOR PCR system.

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Monitoring of Inhibitors of Enzymatic Amplification in Polymerase Chain Reaction and Evaluation of Efficacy of RNA Extraction for the Detection of Hepatitis C Virus Using the Internal Control

Cited by 14 publications

References 13 publications

Screening of blood from potential organ and cornea donors for viruses

Screening of blood from potential organ and cornea donors for viruses

Association between oral anticoagulants and COVID-19 related outcomes: two cohort studies

Automated Specific Capture of Hepatitis C Virus RNA with Probes and Paramagnetic Particle Separation

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