Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

AbstractBackgroundLarge-scale testing for SARS-CoV-2 by RT-PCR is a key element of the response to COVID-19, but little attention has been paid to the potential frequency and impacts of false positives.MethodsFrom a meta-analysis of external quality assessments of RT-PCR assays of RNA viruses, we derived a conservative estimate of the range of false positive rates that can reasonably be expected in SARS-CoV-2 testing, and analyzed the effect of such rates on analyses of regional test data and estimates of population prevalence and asymptomatic ratio.FindingsReview of external quality assessments revealed false positive rates of 0-16.7%, with an interquartile range of 0.8-4.0%. Such rates would have large impacts on test data when prevalence is low. Inclusion of such rates significantly alters four published analyses of population prevalence and asymptomatic ratio.InterpretationThe high false discovery rate that results, when prevalence is low, from false positive rates typical of RT-PCR assays of RNA viruses raises questions about the usefulness of mass testing; and indicates that across a broad range of likely prevalences, positive test results are more likely to be wrong than are negative results, contrary to public health advice about SARS-CoV-2 testing. There are myriad clinical and case management implications. Failure to appreciate the potential frequency of false positives and the consequent unreliability of positive test results across a range of scenarios could unnecessarily remove critical workers from service, expose uninfected individuals to greater risk of infection, delay or impede appropriate medical treatment, lead to inappropriate treatment, degrade patient care, waste personal protective equipment, waste human resources in unnecessary contact tracing, hinder the development of clinical improvements, and weaken clinical trials. Measures to raise awareness of false positives, reduce their frequency, and mitigate their effects should be considered.

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Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript

Journal of Virological Methods ◽

10.1016/j.jviromet.2011.05.017 ◽

2011 ◽

Vol 176 (1-2) ◽

pp. 9-13 ◽

Cited By ~ 16

Author(s):

Helen Auburn ◽

Mark Zuckerman ◽

Simon Broughton ◽

Anne Greenough ◽

Melvyn Smith

Keyword(s):

Internal Control ◽

Rna Viruses ◽

Rt Pcr ◽

Pcr Assays

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Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-021-04159-9 ◽

2021 ◽

Author(s):

Ute Eberle ◽

◽

Clara Wimmer ◽

Ingrid Huber ◽

Antonie Neubauer-Juric ◽

...

Keyword(s):

Real Time ◽

Rt Pcr ◽

Diagnostic Assays ◽

Molecular Assays ◽

Pcr Assays ◽

Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

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Prolonged SARS-CoV-2-RNA Detection from Nasopharyngeal Swabs in an Oncologic Patient: What Impact on Cancer Treatment?

Current Oncology ◽

10.3390/curroncol28010083 ◽

2021 ◽

Vol 28 (1) ◽

pp. 847-852

Author(s):

Anna Ferrari ◽

Marco Trevenzoli ◽

Lolita Sasset ◽

Elisabetta Di Liso ◽

Toni Tavian ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Treatment ◽

Cancer Patients ◽

Clinical Symptoms ◽

Palliative Radiotherapy ◽

Rt Pcr ◽

Cancer Treatments ◽

Global Challenge ◽

Viral Culture ◽

Pcr Assays

The pandemic of SARS-CoV-2 is a serious global challenge affecting millions of people worldwide. Cancer patients are at risk for infection exposure and serious complications. A prompt diagnosis of SARS-CoV-2 infection is crucial for the timely adoption of isolation measures and the appropriate management of cancer treatments. In lung cancer patients the symptoms of infection 19 may resemble those exhibited by the underlying oncologic condition, possibly leading to diagnostic overlap and delays. Moreover, cancer patients might display a prolonged positivity of nasopharyngeal RT-PCR assays for SARS-CoV-2, causing long interruptions or delay of cancer treatments. However, the association between the positivity of RT-PCR assays and the patient’s infectivity remains uncertain. We describe the case of a patient with non-small cell lung cancer, and a severe ab extrinseco compression of the trachea, whose palliative radiotherapy was delayed because of the prolonged positivity of nasopharyngeal swabs for SARS-CoV-2. The patient did not show clinical symptoms suggestive of active infection, but the persistent positivity of RT-PCR assays imposed the continuation of isolation measures and the delay of radiotherapy for over two months. Finally, the negative result of SARS-CoV-2 viral culture allowed us to verify the absence of viral activity and to rule out the infectivity of the patient, who could finally continue her cancer treatment.

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Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽

10.1159/000517003 ◽

2021 ◽

pp. 1-6

Author(s):

Salman Khan ◽

Syed Asad Ali Shah ◽

Syed Muhammad Jamal

Keyword(s):

Polymerase Chain Reaction ◽

Viral Genome ◽

Foot And Mouth Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Chain Reaction ◽

Kappa Value ◽

Polymerase Chain ◽

Pcr Assays ◽

Level Of Agreement

Background: Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. Methods: A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. Results: S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (p = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. Conclusions: The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

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Quantitation of replication of the HCV genome in human livers with end-stage cirrhosis by strand-specific real-time RT-PCR assays: Methods and clinical relevance

Journal of Medical Virology ◽

10.1002/jmv.21510 ◽

2009 ◽

Vol 81 (9) ◽

pp. 1569-1575 ◽

Cited By ~ 4

Author(s):

Lan Lin ◽

Louis Libbrecht ◽

Jannick Verbeeck ◽

Chris Verslype ◽

Tania Roskams ◽

...

Keyword(s):

Real Time ◽

Clinical Relevance ◽

Rt Pcr ◽

Pcr Assays ◽

Human Livers ◽

End Stage

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Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus

Journal of Virological Methods ◽

10.1016/j.jviromet.2014.02.015 ◽

2014 ◽

Vol 201 ◽

pp. 79-85 ◽

Cited By ~ 9

Author(s):

Michele Drigo ◽

Giovanni Franzo ◽

Ilaria Belfanti ◽

Marco Martini ◽

Alessandra Mondin ◽

...

Keyword(s):

Real Time ◽

Respiratory Syndrome Virus ◽

Rt Pcr ◽

End Point ◽

Pcr Assays

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Comparison of ELISA and RT-PCR assays for the detection of Tomato spotted wilt virus in peanut

Peanut Science ◽

10.3146/ps08-024.1 ◽

2009 ◽

Vol 36 (2) ◽

pp. 133-137 ◽

Cited By ~ 1

Author(s):

P. M. Dang ◽

D. L. Rowland ◽

W. H. Faircloth

Keyword(s):

Tomato Spotted Wilt Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Infection Rates ◽

Rt Pcr ◽

Chi Square ◽

Tomato Spotted Wilt ◽

Significant Difference ◽

Pcr Assays ◽

Chi Square Analysis ◽

Wilt Virus

Abstract Diagnosis of Tomato spotted wilt virus (TSWV) in peanut can be accomplished by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction (RT-PCR) but there has been no report of a direct comparison of the success of the two assays in evaluating infection rates of field-grown peanut. We collected peanut root samples from field-grown plants, 76 in 2006 and 48 in 2007, and tested these samples by both ELISA and RT-PCR assays for the presence of TSWV. Out of 124 samples, 50 (40.3%) and 57 (46.0%) were positive for TSWV by ELISA and RT-PCR respectively. In 13.7% of these samples, ELISA and RT-PCR differed in their results. However, Chi square analysis showed no significant difference between the results for these two assays. This result supports the conclusion that ELISA and RT-PCR are comparable for detecting TSWV infection rates in field-grown peanuts.

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