scholarly journals Approbation of RT-qPCR test kit for differential diagnosis of African and Classical swine fever

2018 ◽  
Vol 20 (83) ◽  
pp. 221-225
Author(s):  
S.S. Mandyhra ◽  
L.M. Muzykina ◽  
L.M. Ishchenko ◽  
G.A. Kovalenko ◽  
I.V. Halka ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
Veterinary Medicine ◽  
Internal Control ◽  
Limit Of Detection ◽  
Control Sample ◽  
Classical Swine Fever ◽  
Analytical Sensitivity ◽  
Test Kit ◽  
Field Samples ◽  
Sensitivity Specificity

The first Ukrainian real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) based test kit for the differential diagnosis of African (AFS) and Classical swine fever (CSF) has been developed in the Institute of Veterinary Medicine of NAAS. The proposed test kit allows simultaneous detection of three targets: ASFV DNA, CSFV cDNA and an internal control sample. The goal of this work was to provide an expert evaluation of the RT-qPCR kit for differential diagnosis of ASF and CSF according to appearance, analytical sensitivity, specificity and repeatability. Interdepartmental evaluation of the kit was conducted in the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (DNDILDEVSE) in accordance with the approved methodology. The RT-qPCR kit sensitivity was determined by testing 10-fold serial dilutions of the ASFV DNA and CSFV cDNA (concentration range was 103–100 copies/μl). For specificity determination reference samples of ASFV DNA different genotypes, ASF and CSF positive and negative field samples, as well as pathogens which cause similar to ASF and CSF clinical syndromes were used. Sample preparation and amplification were performed according to the test kit instructions. The amplification was accomplished on QuantStudio™ 5 System (Applied Biosystems). As a result of accomplished interdepartmental evaluation high sensitivity, specificity and repeatability of RT-qPCR kit were confirmed. In particular, it was determined that the limit of detection of the RT-qPCR kit was 5 copies of the ASFV and CSFV genomes per one reaction. The high specificity of the assay to ASFV (I, II, V, VIII, IX and X genotypes) and CSFV was confirmed. It was showп no cross-reactions with closely related pigs viruses (porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and virus of Aujeszky's disease). The high enough repeatability of results was also confirmed. In conclusion, the obtained results are in compliance with the requirements of the RT-qPCR kit normative and technical documentation. This RT-qPCR kit will be recommended for use in veterinary medicine laboratories after its registration would be done.

scholarly journals Detection of EGFR deletion using unique RepSeq technology

2019 ◽  
Author(s):  
Thuraiayah Vinayagamoorthy ◽  
Dahui Qin ◽  
Fei Ye ◽  
Minghao Zhong
Keyword(s):  
Internal Control ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Wild Type ◽  
Sequencing Technology ◽  
Sequencing Method ◽  
Exon 19 Deletion ◽  
Sensitivity Specificity ◽  
Generation Sequencing

AbstractWe are reporting a novel sequencing technology, RepSeq (Repetitive Sequence), that has high sensitivity, specificity and quick turn-around time. This new sequencing technology is developed by modifying traditional Sanger sequencing technology in several aspects. The first, a homopolymer tail is added to the PCR primer(s), which makes interpreting electropherograms a lot easier than that in traditional Sanger sequencing. The second, an indicator nucleotide is added at the 5’end of the homopolymer tail. In the presence of a deletion, the position of the indicator nucleotide in relation to the wild type confirms the deletion. At the same time, the indicator of the wild type serves as the internal control. Furthermore, the specific design of the PCR and/or sequencing primers will specifically enrich/select mutant alleles, which increases sensitivity and specificity significantly. Based on serial dilution studies, the analytical lower limit of detection was 1.47 copies. A total of 89 samples were tested for EGFR exon 19 deletion, of which 21 were normal blood samples and 68 were samples previously tested by either pyrosequencing or TruSeq Next Generation Sequencing Cancer Panel. There was 100 % concordance among all the samples tested. RepSeq technology has overcome the shortcomings of Sanger sequencing and offers an easy-to-use novel sequencing method for personalized precision medicine.

scholarly journals Polymethacrylate Sphere-Based Assay for Ultrasensitive miRNA Detection

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Samira Hosseini ◽  
Patricia Vázquez-Villegas ◽  
Richard C. Willson ◽  
Marco Rito-Palomares ◽  
Margarita Sanchez-Dominguez ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Breast Cancer Incidence ◽  
High Specific Surface Area ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Important Target ◽  
Target Analytes ◽  
High Sequence Homology ◽  
Mirna Detection ◽  
Sensitivity Specificity

Although microRNAs (miRNAs) have emerged as increasingly important target analytes, their biorecognition remains challenging due to their small size, high sequence homology, and low abundance in clinical samples. Nanospheres and microspheres have also gained increasing attention in biosensor applications due to their high specific surface area and the wide variety of compositions available. In this study, chemically designed and synthesized microspheres with active functional groups were used to promote effective miRNA immobilization resulting in better biorecognition. Upon conjugation with fluorescence-labeled complimentary probes, acylate-based spheres have indirectly detected MiR159, offering significantly enhanced analytical sensitivity, specificity, and accuracy while yielding a considerably low limit of detection (LOD) of 40 picomolar. Furthermore, MiR159 presence, which is known to be inversely correlated to breast cancer incidence and progression, was successfully detected in a competitive assay, which is promising for upgrading the current assay to clinical use.

scholarly journals Pathogenic and Virulence Factor Detection on Viable but Non-culturable Methicillin-Resistant Staphylococcus aureus

2021 ◽  
Vol 12 ◽  
Author(s):  
Hua Jiang ◽  
Kan Wang ◽  
Muxia Yan ◽  
Qian Ye ◽  
Xiaojing Lin ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Limit Of Detection ◽  
Foodborne Pathogen ◽  
Food Poisoning ◽  
Clinical Settings ◽  
Analytical Sensitivity ◽  
Vbnc State ◽  
Methicillin Resistant ◽  
Staphylococcal Enterotoxins ◽  
Sensitivity Specificity

Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called “standards,” “guidelines,” or “gold standards” are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.

scholarly journals Commercial stocks of SARS-CoV-2 RNA may report low concentration values, leading to artificially increased apparent sensitivity of diagnostic assays

2020 ◽  
Author(s):  
Erik Jue ◽  
Rustem F. Ismagilov
Keyword(s):  
Diagnostic Test ◽  
Single Molecule ◽  
Reverse Transcription ◽  
Plasmid Dna ◽  
Limit Of Detection ◽  
Analytical Sensitivity ◽  
Genomic Rna ◽  
Actual Concentration ◽  
Test Kit

AbstractIn response to the rapidly evolving COVID-19 pandemic, the U.S. Food and Drug Administration (FDA) has rapidly issued 49 emergency use authorizations (EUAs) for SARS-CoV-2 in vitro diagnostic test-kits. A critical metric in the performance evaluation for a diagnostic test kit is the analytical sensitivity, which is measured by the limit of detection (LOD). Commercial RNA stocks with known titers are used to determine LOD. We identified a problem with the titer reported for the commercial stocks when examining the analytical sensitivity of the reverse transcription quantitative PCR (RT-qPCR) protocol that is recommended by the Centers for Disease Control and Prevention (CDC) using plasmid DNA from Integrated DNA Technologies (IDT), synthetic RNA from BEI Resources (BEI), and extracted genomic RNA from BEI. We detected 3/3 positives for reactions containing synthetic RNA at a concentration of 0.1 copies/reaction (based on the supplier’s label concentration). The apparent better-than-single-molecule performance is a statistically highly unlikely event, indicating a potential inaccuracy in the supplier’s quantification of the stock material. Using an ultrasensitive and precise assay, reverse transcription digital PCR (RT-dPCR), we independently quantified concentrations of commercial SARS-CoV-2 plasmid DNA and SARS-CoV-2 RNA stocks. For plasmid DNA, the actual concentration measured by RT-dPCR was 11% of the nominal label concentration. For synthetic RNA, the actual concentration measured by RT-dPCR for one lot was 770% of the label concentration and for a different lot was 57% of the label concentration. For genomic RNA, the concentration measured by RT-dPCR for one lot was 240% of the label concentration and for a different lot it was 300% of the label concentration. This SARS-CoV-2 genomic RNA from BEI Resources has been used in at least 11 approved FDA Emergency Use Authorizations as of April 27, 2020. Such deviations of reported RNA or DNA stock concentrations from true concentrations can result in inaccurate quantification and calculation of LOD. Precise and accurate reporting of DNA and RNA stock concentrations by commercial suppliers will enable accurate quantification of assay performance, which is urgently needed to improve evaluation of different assays by diagnostic developers and regulatory bodies.

scholarly journals Accuracy of the Mologic COVID-19 rapid antigen test: a prospective multi-centre analytical and clinical evaluation

2021 ◽  
Vol 6 ◽  
pp. 132
Author(s):  
Ana I Cubas-Atienzar ◽  
Fiona Bell ◽  
Rachel L. Byrne ◽  
Kate Buist ◽  
David J. Clark ◽  
...  
Keyword(s):  
Clinical Presentation ◽  
Limit Of Detection ◽  
Sampling Strategy ◽  
Cross Reactivity ◽  
Analytical Sensitivity ◽  
Antigen Test ◽  
Point Of Use ◽  
Clinical Accuracy ◽  
Polymerase Chain ◽  
Sensitivity Specificity

Background: The coronavirus disease 2019 (COVID-19) pandemic has highlighted the reliance on antigen detection rapid diagnostic tests (Ag-RDTs). Their evaluation at point of use is a priority. Methods: Here, we report a multi-centre evaluation of the analytical sensitivity, specificity, and clinical accuracy of the Mologic COVID-19 Ag-RDT by comparing to reverse transcriptase polymerase chain reaction (RT-qPCR) results from individuals with and without COVID-19 symptoms. Participants had attended hospitals in Merseyside, hospital and ambulance services in Yorkshire, and drive-through testing facilities in Northumberland, UK. Results: The limit of detection of the Mologic COVID-19 Ag-RDT was 5.0 x 102 pfu/ml in swab matrix with no cross-reactivity and interference for any other pathogens tested. A total of 347 participants were enrolled from 26th of November 2020 to 15th of February 2021 with 39.2% (CI 34.0-44.6) testing RT-qPCR positive for SARS-CoV-2. The overall sensitivity and specificity of the Mologic Ag-RDT compared to the reference SARS-CoV-2 RT-qPCR were 85.0% (95% CI 78.3-90.2) and 97.8% (95.0-99.3), respectively. Sensitivity was stratified by RT-qPCR cycle threshold (Ct) and 98.4% (91.3-100) of samples with a Ct less than 20 and 93.2% (86.5-97.2) of samples with a Ct less than 25 were detected using the Ag-RDT. Clinical accuracy was stratified by sampling strategy, swab type and clinical presentation. Mologic COVID-19 Ag-RDT demonstrated highest sensitivity with nose/throat swabs compared with throat or nose swabs alone; however, the differences were not statistically significant. Conclusions: Overall, the Mologic test had high diagnostic accuracy across multiple different settings, different demographics, and on self-collected swab specimens. These findings suggest the Mologic rapid antigen test may be deployed effectively across a range of use settings.

FUZZY MODELS OF DIFFERENTIAL DIAGNOSIS OF SEROUS AND PURULENT PYELONEPHRITIS IN PATIENTS WITH UROLITHIASIS

2021 ◽  
pp. 106-113
Author(s):  
Станислав Петрович Серегин ◽  
Дмитрий Сергеевич Родионов ◽  
Наталья Анатольевна Милостная ◽  
Геннадий Вячеславович Сипливый ◽  
Роман Анатольевич Крупчатников
Keyword(s):  
Differential Diagnosis ◽  
Decision Rules ◽  
Control Sample ◽  
Fuzzy Rules ◽  
Similar Data ◽  
Recognition Theory ◽  
Therapeutic Measures ◽  
Sensitivity Specificity ◽  
High Degree

В настоящее время все большее распространение имеет проблема возникновения мочекаменной болезни, в том числе отягощенная осложнениями, такими как серозный и гнойный пиелонефрит. Задача диагностики различных форм пиелонефрита относится к классу плохоформализуемых задач, которые недостаточно эффективно решаются классическими методами теории распознавания образов. Практика решения задач с аналогичной структурой данных показала, что приемлемое для практики качество принятия решений по поставленной в работе задачи может быть достигнуто при использовании методологии синтеза гибридных нечетких правил. В связи с этим, целью исследования является повышение качества дифференциальной диагностики серозного и гнойного пиелонефрита у больных мочекаменной болезнью на основе данных оксидантного статуса с использованием названной методологии реализуемой современными информационными и интеллектуальными технологиями. Для достижения поставленной в работе цели, в соответствии с общими рекомендациями методологии синтеза гибридных нечетких правил была синтезирована система гибридных диагностических моделей на основе модифицированной формулы Е. Шортлифа, в которой коэффициент уверенности от одного признака заменяется соответствующей функцией принадлежности. Полученная система решающих правил, позволяет с высокой степенью уверенности производить дифференциальную диагностику серозных и гнойных форм пиелонефрита с выделением промежуточного класса состояний, требующего проведения специфических терапевтических мероприятий. Проверка результатов срабатывания полученных решающих правил на репрезентативной контрольной выборке показала, что диагностические чувствительность, специфичность и эффективность предлагаемого метода не ниже 0,9, что позволяет рекомендовать полученные результаты для использования в медицинской практике Currently, the problem of urolithiasis is becoming more common, including complications such as serous and purulent pyelonephritis. The problem of diagnosing various forms of pyelonephritis belongs to the class of poorly formalized problems that are not effectively solved by classical methods of pattern recognition theory. The practice of solving problems with a similar data structure has shown that an acceptable quality of decision-making for the problem set in the work can be achieved using the methodology of hybrid fuzzy rules synthesis. In this regard, the aim of the study is to improve the quality of differential diagnosis of serous and purulent pyelonephritis in patients with urolithiasis based on the data of the oxidant status using the named methodology implemented by modern information and intelligent technologies. To achieve this goal, in accordance with the general recommendations of the methodology for the synthesis of hybrid fuzzy rules, a system of hybrid diagnostic models was synthesized based on the modified E. Shortlif formula, in which the confidence coefficient from one attribute is replaced by the corresponding membership function. The resulting system of decisive rules allows for a high degree of confidence in the differential diagnosis of serous and purulent forms of pyelonephritis with the allocation of an intermediate class of conditions that require specific therapeutic measures. Verification of the results of the operation of the obtained decision rules on a representative control sample showed that the diagnostic sensitivity, specificity and effectiveness of the proposed method is not lower than 0.9, which allows us to recommend the results for use in medical practice

scholarly journals Evaluation of a Commercial Competitive ELISA Test Kit for the Detection of Group-Specific Antibodies to Bluetongue Virus

1993 ◽  
Vol 5 (3) ◽  
pp. 336-340 ◽  
Author(s):  
A. Afshar ◽  
H. C. Trotter ◽  
G. C. Dulac ◽  
J. J. Reddington
Keyword(s):  
Bluetongue Virus ◽  
Elisa Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Analytical Sensitivity ◽  
Serum Samples ◽  
Specific Antibodies ◽  
Test Kit ◽  
Field Samples ◽  
The Usa ◽  
Haemorrhagic Disease

The performance of a competitive (c) enzyme-linked immunosorbent assay (ELISA) test kit, Blueplate Special@ (BPS), commercially produced by DiagXotics (Wilton, CT) for detection of group-specific antibodies to bluetongue virus (BTV) was compared with that of an internationally endorsed cELISA-I. A total of 1,026 serum samples were tested in this study: 133 samples from 23 calves and 3 sheep experimentally infected with South African isolates of 19 BTV serotypes and US isolates of 5 BTV serotypes and 7 calves infected with US isolates of 2 epizootic haemorrhagic disease of deer virus (EHDV); 102 paired sera from cattle, sheep, and goats experimentally infected with the Australian isolates of BTV, EHDV, and Palyam virus; 229 bovine and ovine samples of Canadian origin (BTV free); and 562 bovine and ovine field samples from the USA and Barbados (BTV endemic). Seroconversion was demonstrable by the BPS cELISA 10 days postinfection in all experimental animals inoculated with BTV, with the exception of 4 calves in which there was a delay of 10–20 days. Similar to the cELISA-I, none of the sera from calves inoculated with US and Australian isolates of EHDV and Palyam viruses cross-reacted with the BTV antigen in the BPS cELISA. The total agreement between the two assays for all the total bovine and ovine field sera was 98.1%. The overall results substantiate the usefulness of the BPS cELISA test kit for monitoring animal sera for group-specific antibodies to BTV. The slightly lower analytical sensitivity associated with the detection of antibody during early phase of infection in some animals would not be significant in the context of herd testing or any regulatory program.

scholarly journals Clinical Performance and Analytical Sensitivity of Three SARS-CoV-2 Nucleic Acid Diagnostic Tests

2021 ◽  
Vol 104 (4) ◽  
pp. 1516-1518
Author(s):  
Byron Freire-Paspuel ◽  
Miguel Angel Garcia-Bereguiain
Keyword(s):  
Nucleic Acid ◽  
Gold Standard ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Analytical Sensitivity ◽  
Nucleic Acid Test ◽  
Test Kit ◽  
Food Drug Administration ◽  
Made In China ◽  
Acid Test

ABSTRACTHundreds of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with emergency use authorization (EUA) by the Food Drug Administration (FDA) or their country of origin agency, but also many of them without any independent clinical performance evaluation. We performed a clinical evaluation for two Chinese SARS-CoV-2 RT-PCR kits available in South America, COVID-19 Nucleic Acid Test Kit (eDiagnosis Biomedicine, Wuhan, China) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech, Changsha, China), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found an excellent clinical performance and analytical sensitivity for both kits with sensitivity values of 100% and 95.3% and estimated limit of detection of 500 copies/mL and 1,000 copies/mL, for eDiagnosis and Sansure Biotech kits, respectively. COVID-19 Nucleic Acid Test Kit (eDiagnosis) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech) are both made in China and hold EUA by the Chinese CDC. Also, Sansure Biotech kit has EUA by the FDA. In conclusion, our results endorse the use of these two commercially available kits imported to Ecuador for SARS-CoV-2 diagnosis, as they had the similar clinical performance as the gold standard from the CDC.

Detectability of Plasma Proteins in SRM Measurements

2018 ◽  
Vol 16 (1) ◽  
pp. 74-81 ◽  
Author(s):  
Olga I. Kiseleva ◽  
Elena A. Ponomarenko ◽  
Yulia A. Romashova ◽  
Ekaterina V. Poverennaya ◽  
Andrey V. Lisitsa
Keyword(s):  
Blood Plasma ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Human Blood Plasma ◽  
Analytical Sensitivity ◽  
Plasma Proteome ◽  
Protein Targets ◽  
Blood Plasma Sample ◽  
Specificity And Sensitivity ◽  
Human Plasma Proteome

Background: Liquid chromatography coupled with targeted mass spectrometry underwent rapid technical evolution during last years and has become widely used technology in clinical laboratories. It offers confident specificity and sensitivity superior to those of traditional immunoassays. However, due to controversial reports on reproducibility of SRM measurements, the prospects of clinical appliance of the method are worth discussing. </P><P> Objective: The study was aimed at assessment of capabilities of SRM to achieve a thorough assembly of the human plasma proteome. </P><P> Method: We examined set of 19 human blood plasma samples to measure 100 proteins, including FDA-approved biomarkers, via SRM-assay. </P><P> Results: Out of 100 target proteins 43 proteins were confidently detected in at least two blood plasma sample runs, 36 and 21 proteins were either not detected in any run or inconsistently detected, respectively. Empiric dependences on protein detectability were derived to predict the number of biological samples required to detect with certainty a diagnostically relevant quantum of the human plasma proteome. </P><P> Conclusion: The number of samples exponentially increases with an increase in the number of protein targets, while proportionally decreasing to the logarithm of the limit of detection. Analytical sensitivity and enormous proteome heterogeneity are major bottlenecks of the human proteome exploration.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

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