scholarly journals Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results

2021 ◽  
pp. 1-18
Author(s):  
Jonathan B. Gubbay ◽  
Heather Rilkoff ◽  
Heather L. Kristjanson ◽  
Jessica D. Forbes ◽  
Michelle Murti ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Real Time ◽  
Reverse Transcription ◽  
Predictive Value ◽  
Large Scale ◽  
Rdrp Gene ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Test Probability

Abstract Objectives Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false positive test. This study assessed positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Methods A total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. Results Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤ 3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the four groups with higher pre-test probability (individual group range 50·0% to 85·0%). Conclusions Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

scholarly journals Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results

2021 ◽  
Author(s):  
Jonathan B. Gubbay ◽  
Heather Rilkoff ◽  
Heather L. Kristjanson ◽  
Jessica D. Forbes ◽  
Michelle Murti ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
False Positive ◽  
Rdrp Gene ◽  
Positive Test ◽  
Test Results ◽  
Rt Pcr ◽  
Pcr Assay ◽  
False Positive Test ◽  
Test Probability

ABSTRACTBackgroundPerformance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent resolved infection with persistent RNA shedding, presymptomatic or asymptomatic infection, or a false positive test. This study assessed clinical specificity of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay.Materials and MethodsA total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing.ResultsSignificantly less positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤ 3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in all other groups combined (5/32, 15·6% vs 61/90, 68%; p <0·0001), and in each subgroup with higher pre-test probability (individual subgroup range 50·0% to 85·0%).ConclusionsA higher proportion of false-positive test results are likely with lower pre-test probability. Positive SARS-CoV-2 PCR results should be interpreted within the context of patient history, clinical setting, known exposure, and estimated community disease prevalence. Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea

2004 ◽  
Vol 72 (3) ◽  
pp. 496-501 ◽  
Author(s):  
Xiaoli L. Pang ◽  
Bonita Lee ◽  
Nasim Boroumand ◽  
Barbara Leblanc ◽  
Jutta K. Preiksaitis ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Stool Specimens ◽  
Polymerase Chain

Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (5) ◽  
pp. 840-843 ◽  
Author(s):  
AYSUN YILMAZ ◽  
KAMIL BOSTAN ◽  
EDA ALTAN ◽  
KARLO MURATOGLU ◽  
NURI TURAN ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Epidemiological Studies ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Food Items ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Tomato Sample ◽  
Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

scholarly journals Comparative Assessment of Sensitivity and Specificity of Rose Bengal Test and Modified In-house ELISA by using IS711 TaqMan Real Time PCR Assay as a Gold Standard for the Diagnosis of Bovine Brucellosis

2018 ◽  
Vol 11 (2) ◽  
pp. 951-957
Author(s):  
Amira M. Zakaria
Keyword(s):  
Positive Predictive Value ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Rose Bengal ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Gold Standard ◽  
Bovine Brucellosis ◽  
Rt Pcr ◽  
Rose Bengal Test

Serological approaches such as Rose Bengal Test (RBT) and enzyme linked immunosorbent assay (ELISA) are common tests, however they are generally not sensitive or specific enough for diagnosis of Brucella because of cross-reactivity with different bacterial antigens. The work objected to evaluate the sensitivity and specificity of rose Bengal and modified in-house ELISA using IS711 real time PCR as a gold test to detect Brucella in calves sera. Two hundred and thirty (n=230) blood samples were collected from (2-3) years asymptomatic male calves in two Egyptian abattoirs. Rose Bengal test (RBT) and modified in-house ELISA were applied to determine the seroprevalence of brucellosis in abattoirs animals while quantitative Taqman real-time PCRs (RT-PCR) were implemented for the identification of Brucella genus. The overall prevalence of brucellosis was (53.9 %), (75.2 %) and (79.1 %) as determined by ELISA,RBT and RT- PCR assays respectively. Regarding statistical analysis of the reported data and considering real time PCR the gold standard, the RBT recorded a sensitivity of (79.12%) and a specificity of (39.58 %) with an accuracy of (70.87%). While (83.24%) was reported as positive predictive value and (33.33 %) as a negative predictive value. The sensitivity and specificity of ELISA were (55.49%) and (52.08 %) respectively while the accuracy was (54.78%). Positive predictive value and negative predictive value for ELISA were determined as (81.45%) and (23.58 %) respectively. RBT can be routinely used for diagnosis of Brucella as cost effective , more sensitive and accurate test than ELISA However, real time PCR is highly recommend as gold test for identification and differentiation of bovine brucellosis.

scholarly journals Performance of Real-Time Reverse Transcription Polymerase Chain Reaction for the Detection of Foot-and-Mouth Disease Virus during Field Outbreaks in the United Kingdom in 2007

2009 ◽  
Vol 21 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Scott M. Reid ◽  
Katja Ebert ◽  
Katarzyna Bachanek-Bankowska ◽  
Carrie Batten ◽  
Anna Sanders ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
United Kingdom ◽  
Real Time ◽  
Reverse Transcription ◽  
Foot And Mouth Disease ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The United Kingdom ◽  
Polymerase Chain

Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). The present report describes the practical steps undertaken to deploy a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to process the samples received during the outbreaks of FMD in the United Kingdom in 2007. Two independent real-time RT-PCR assays targeting different regions (5′UTR and 3D) of the FMD virus (FMDV) genome were used to confirm the presence of FMDV in clinical samples collected from the first infected premises. Once the FMDV strain responsible had been sequenced, a single real-time RT-PCR assay (3D) was selected to test a total of 3,216 samples, including material from all 8 infected premises. Using a 96-well automated system to prepare nucleic acid template, up to 84 samples could be processed within 5 hr of submission, and up to 269 samples were tested per working day. A conservative cut-off was used to designate positive samples, giving rise to an assay specificity of 99.9% or 100% for negative control material or samples collected from negative premises, respectively. For the first time, real-time RT-PCR results were used to recognize preclinical FMD in a cattle herd. Furthermore, during the later stages of the outbreaks, the real-time RT-PCR assay supported an active surveillance program within high-risk cattle herds. To the authors' knowledge, this is the first documented use of real-time RT-PCR as a principal laboratory diagnostic tool following introduction of FMD into a country that was FMD-free (without vaccination) and highlights the advantages of this assay to support control decisions during disease outbreaks.

Real-time reverse transcription-polymerase chain reaction for detection of SYT-SSX translocation in synovial sarcoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 9553-9553
Author(s):  
J. Liu ◽  
K. Qu ◽  
C. Chai ◽  
H. Li ◽  
A. Sferruzza ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Synovial Sarcoma ◽  
Internal Control ◽  
Reverse Transcription ◽  
Gel Electrophoresis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

9553 Background: Synovial sarcoma is the most common non-rhabdomyosarcomatous soft tissue sarcoma in children and adolescents. A specific translocation, t(X;18), induces fusion of the SYT gene on chromosome 18 to an SSX gene on chromosome X. The resulting fusion gene consists of at least 2 subtypes with different breakpoints: SYT-SSX1(X;18)(p11.23;q11.2) and SYT-SSX2 (X;18)(p11.21;q11.2). Because t(X;18) transcripts occur in >90% of synovial sarcoma subtypes, this marker may be useful for diagnosis. We evaluated the accuracy of a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of the primary SYT-SSX fusion transcript types in formalin-fixed, paraffin-embedded (FFPE) tissues and frozen tissues. Methods: 17 tumors (7 synovial sarcomas, 4 Ewing’s sarcomas, 5 rhabdomyosarcomas, 1 small round blue-cell tumor), 4 normal tissues, and 4 control samples were tested for SYT-SSX translocations using real-time RT-PCR. Results were compared to those obtained with gel electrophoresis detection of amplified transcripts; discrepant results were confirmed by sequencing. Results: Concordance between real time RT-PCR and gel electrophoresis was 100% (25/25) for internal control genes and SYT-SSX1, and 92% (23/25) for SYT-SSX2. Of the 2 samples with discordant SYT-SSX2 results, 1 was positive by real-time RT-PCR but not gel electrophoresis and 1 was positive by electrophoresis but not real-time RT-PCR; in both cases, DNA sequencing confirmed the real-time RT-PCR results. The minimum percentage of tumor to normal cells required for detection of SYT-SSX fusion transcripts by real-time RT-PCR was 6.25%. Conclusions: This real-time RT-PCR assay appears to provide greater accuracy than gel electrophoresis for identification of SYT-SSX translocation and fusion types. No significant financial relationships to disclose.

scholarly journals Quantitative Real-Time Reverse Transcription-PCR Assay for Cyclin D1 Expression: Utility in the Diagnosis of Mantle Cell Lymphoma

2001 ◽  
Vol 47 (2) ◽  
pp. 195-201 ◽  
Author(s):  
Karen E Bijwaard ◽  
Nadine S I Aguilera ◽  
Yury Monczak ◽  
Michel Trudel ◽  
Jeffery K Taubenberger ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Cyclin D1 ◽  
Real Time ◽  
Reverse Transcription ◽  
Detection System ◽  
Regulatory Elements ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Sequence Detection ◽  
Mantle Cell

Abstract Background: The t(11;14)(q13;q32) translocation present in the majority of mantle cell lymphomas (MCLs) places the cyclin D1 gene under the control of immunoglobulin transcriptional regulatory elements, causing overexpression of cyclin D1. Quantification of cyclin D1 expression can distinguish MCL from other lymphomas. Methods: A quantitative real-time reverse transcription (RT)-PCR assay was developed for cyclin D1 mRNA suitable for use with RNA extracted from fresh and formalin-fixed, paraffin-embedded tissues. Specimens were amplified in an Applied Biosystems Model 7700 Sequence Detection System in reactions containing primers and probes for cyclin D1 and a control gene, β2-microglobulin. Relative expression of the two genes was standardized against a control MCL cell line, M02058. Results: The range of cyclin D1 expression among 20 MCLs was substantially higher than that in other lymphomas and reactive lymph nodes. By choosing an optimal cutoff point for assessing overexpression, the sensitivity and specificity of the assay for the diagnosis of MCL in lymph node specimens both approached 100%: Overexpression was detected in 20 of 20 MCLs, but in none of 21 non-mantle-cell lymphomas or 10 reactive lymph nodes. Conclusions: Quantitative real-time RT-PCR for cyclin D1 overexpression provides a rapid diagnostic test with clinical utility in the diagnosis of MCL.

scholarly journals Combined Immunomagnetic Separation-Molecular Beacon-Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

2004 ◽  
Vol 70 (7) ◽  
pp. 4371-4374 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Pcr Assay ◽  
The Real

ABSTRACT In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay successfully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water.

scholarly journals Development, Evaluation, and Standardization of a Real-Time TaqMan Reverse Transcription-PCR Assay for Quantification of Hepatitis A Virus in Clinical and Shellfish Samples

2006 ◽  
Vol 72 (6) ◽  
pp. 3846-3855 ◽  
Author(s):  
M. Isabel Costafreda ◽  
Albert Bosch ◽  
Rosa M. Pint�
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Accurate Estimation ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Development Evaluation

ABSTRACT A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5′ noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

scholarly journals Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

2013 ◽  
Vol 79 (21) ◽  
pp. 6585-6592 ◽  
Author(s):  
Takayuki Miura ◽  
Sylvain Parnaudeau ◽  
Marco Grodzki ◽  
Satoshi Okabe ◽  
Robert L. Atmar ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Sewage Sample ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Assay Sensitivity ◽  
Reverse Transcription Pcr ◽  
Specificity And Sensitivity ◽  
Samples Selection

ABSTRACTNorovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.

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