DIFFERENTIAL DIAGNOSIS OF RESPIRATORY VIRUSES BY USING REAL TIME RT-PCR METHODOLOGY

This study aimed at assessing the validity of self-collected (self-sampled) oropharyngeal (OP) swabs among healthcare workers compared to those collected by trained sentinel general practitioners (GP-sampled) from individuals with influenza-like illness (ILI), to be implemented in epidemiological studies and/or surveillance programs of viral pathogens involved in community respiratory infections. In our study, OP swabs were collected from adults (>18 years) with ILI during the 2018–2019 influenza season. Two groups of samples were considered: group 1−131 self-sampled OP swabs collected by healthcare workers after being trained on the sampling procedure; group 2−131 GP-sampled OP swabs collected from outpatients by sentinel GPs operating within the Italian Influenza Surveillance Network. To assess swabbing quality, following RNA extraction, each sample was tested for the presence of the human ribonuclease P gene (RNP) by in-house real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Samples with a cycle threshold (Ct) <35 were considered adequate for further virological analysis. Influenza viruses (IVs), respiratory syncytial virus (RSV), and rhinovirus (RV) genomes were detected by in-house real-time RT-PCR. All samples were positive to RNP detection with Ct <35. The mean Ct value was similar in the two groups (group 1 vs. group 2: 25.93 ± 2.22 vs. 25.46 ± 2.40; p = 0.10). IVs, RSV, and RV positivity rates were 26.7 vs. 52.7% (p < 0.01), 7.6 vs. 9.9% (p = 0.52), and 21.4 vs. 19.9% (p = 0.76), respectively. Self-sampled OP swabs resulted as valid as GP-sampled OP swabs for molecular detection of respiratory viruses. Self-swabbing can thus be a worthwhile strategy for sample collection to implement molecular surveillance of respiratory pathogens and carry out epidemiological studies, easily reaching a larger population size.

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A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses

Journal of Virological Methods ◽

10.1016/j.jviromet.2010.02.005 ◽

2010 ◽

Vol 165 (2) ◽

pp. 254-260 ◽

Cited By ~ 44

Author(s):

R. Lassaunière ◽

T. Kresfelder ◽

M. Venter

Keyword(s):

Real Time ◽

Respiratory Viruses ◽

Rt Pcr ◽

Pcr Assay ◽

Hybridization Probes

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Comparison of Luminex xTAG® RVP fast assay and real time RT-PCR for the detection of respiratory viruses in adults with community-acquired pneumonia

Journal of Medical Virology ◽

10.1002/jmv.24463 ◽

2016 ◽

Vol 88 (7) ◽

pp. 1173-1179 ◽

Cited By ~ 4

Author(s):

Vivian Luchsinger ◽

Yara Prades ◽

Mauricio Ruiz ◽

Rolando Pizarro ◽

Patricio Rossi ◽

...

Keyword(s):

Real Time ◽

Respiratory Viruses ◽

Community Acquired Pneumonia ◽

Rt Pcr

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Detection of Common Respiratory Viruses in Patients with Acute Respiratory Infections Using Multiplex Real-Time RT-PCR

Jundishapur Journal of Microbiology ◽

10.5812/jjm.96513 ◽

2019 ◽

Vol In Press (In Press) ◽

Cited By ~ 1

Author(s):

Niloofar Neisi ◽

Samaneh Abbasi ◽

Manoochehr Makvandi ◽

Shokrollah Salmanzadeh ◽

Somayeh Biparva ◽

...

Keyword(s):

Real Time ◽

Respiratory Infections ◽

Respiratory Viruses ◽

Acute Respiratory Infections ◽

Rt Pcr

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A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses

Virology ◽

10.1016/j.virol.2017.11.021 ◽

2018 ◽

Vol 515 ◽

pp. 29-37 ◽

Cited By ~ 12

Author(s):

Nacira Laamiri ◽

Rim Aouini ◽

Boutheina Marnissi ◽

Abdeljelil Ghram ◽

Issam Hmila

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Respiratory Viruses ◽

Rt Pcr

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Development of a Novel, Genome Subtraction-Derived, SARS-CoV-2-Specific COVID-19-nsp2 Real-Time RT-PCR Assay and Its Evaluation Using Clinical Specimens

International Journal of Molecular Sciences ◽

10.3390/ijms21072574 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2574 ◽

Cited By ~ 29

Author(s):

Cyril Chik-Yan Yip ◽

Chi-Chun Ho ◽

Jasper Fuk-Woo Chan ◽

Kelvin Kai-Wang To ◽

Helen Shuk-Ying Chan ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Respiratory Viruses ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Clinical Specimens ◽

Coronavirus Infection ◽

Polymerase Chain ◽

Novel Coronavirus

The pandemic novel coronavirus infection, Coronavirus Disease 2019 (COVID-19), has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID50/mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed COVID-19-RdRp/Hel reference assay. A rapid, sensitive, SARS-CoV-2-specific real-time RT-PCR assay, COVID-19-nsp2, was developed.

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Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG®RVP fast assay for the detection of respiratory viruses

Journal of Medical Virology ◽

10.1002/jmv.24299 ◽

2015 ◽

Vol 88 (1) ◽

pp. 51-57 ◽

Cited By ~ 17

Author(s):

Manohar L. Choudhary ◽

Siddharth P. Anand ◽

Shamal A. Tikhe ◽

Atul M. Walimbe ◽

Varsha A. Potdar ◽

...

Keyword(s):

Real Time ◽

Respiratory Viruses ◽

Rt Pcr

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Evaluation of a novel real-time RT-PCR using TOCE technology compared with culture and Seeplex RV15 for simultaneous detection of respiratory viruses

Journal of Clinical Virology ◽

10.1016/j.jcv.2013.04.014 ◽

2013 ◽

Vol 57 (4) ◽

pp. 338-342 ◽

Cited By ~ 37

Author(s):

Chi Hyun Cho ◽

Bayarjavkhlan Chulten ◽

Chang Kyu Lee ◽

Myung Hyun Nam ◽

Soo Young Yoon ◽

...

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Respiratory Viruses ◽

Rt Pcr

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Evaluation of the incidence of identified types of respiratory viruses in a selected population of inhabitants of the kuyavian-pomeranian voivodeship

Diagnostyka Laboratoryjna ◽

10.5604/01.3001.0013.7707 ◽

2018 ◽

Vol 54 (3) ◽

pp. 151-158

Author(s):

Małgorzata Smoguła ◽

Marta Pawłowska ◽

Celestyna Mila-Kierzenkowska ◽

Joanna Malinowska ◽

Jerzy Kasprzak ◽

...

Keyword(s):

Influenza Virus ◽

Real Time ◽

Genetic Material ◽

Respiratory Viruses ◽

Influenza B Virus ◽

Influenza B ◽

Rt Pcr ◽

Syncytial Virus ◽

Tract Infections ◽

Epidemiological Situation

Introduction: Respiratory tract infections are caused by various factors including respiratory viruses, for example influenza virus and respiratory syncytial virus (RSV). Surveillance of influenza and other respiratory viruses is carried out as part of the SENTINEL and NON SENTINEL programs in Poland.<br>Aim of study: Determination of the frequency of selected types of influenza and RSV viruses using real-time reverse transcription polymerase chain reaction method (real-time RT-PCR) in a selected population of inhabitants of the kuyavian-pomeranian voivodeship in the epidemic season 2017/2018, i.e. from 1 October 2017 to 30 April 2018.<br>Material and methods: 201 throat and nasal swabs were tested using real-time RT-PCR method for the detection of the presence of genetic material RNA of influenza virus and RSV. The study population was divided into 7 groups depending on age.<br>Results. Positive samples accounted for 48.26% of the swabs tested. The genetic material RNA of influenza virus was found in 95 (47.26) samples and RSV in 2 (1,00%) samples. Influenza B virus was most frequently isolated. The most prevalence of infections was observed in people under 25 years of age.<br>Conclusions: Surveillance of influenza in the SENTINEL and NON SENTINEL programs constitute an important role in assessing the virological and epidemiological situation prevailing in a given area. The results cover part of the population, hence the data may be underestimated. It is necessary to continue studies and observe the virological and epidemiological situation in the polish population.

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Determination of cut-off cycle threshold values in routine RT–PCR assays to assist differential diagnosis of norovirus in children hospitalized for acute gastroenteritis

Epidemiology and Infection ◽

10.1017/s095026881500059x ◽

2015 ◽

Vol 143 (15) ◽

pp. 3292-3299 ◽

Cited By ~ 21

Author(s):

N. V. TRANG ◽

M. CHOISY ◽

T. NAKAGOMI ◽

N. T. M. CHINH ◽

Y. H. DOAN ◽

...

Keyword(s):

Differential Diagnosis ◽

Real Time ◽

Acute Gastroenteritis ◽

Bimodal Distribution ◽

Clinical Samples ◽

Rt Pcr ◽

Prevalence Studies ◽

Cycle Threshold ◽

Asymptomatic Children ◽

Enteric Infections

SUMMARYNorovirus (NV) is an important cause of acute gastroenteritis in children, but is also frequently detected in asymptomatic children, which complicates the interpretation of NV detection results in both the clinical setting and population prevalence studies. A total of 807 faecal samples from children aged <5 years hospitalized for acute gastroenteritis were collected in Thai Binh, Vietnam, from January 2011 to September 2012. Real-time RT–PCR was used to detect and quantify NV-RNA in clinical samples. A bimodal distribution of cycle threshold (Ct) values was observed in which the lower peak was assumed to represent cases for which NV was the causal agent of diarrhoea, whereas the higher peak was assumed to represent cases involving an alternative pathogen other than NV. Under these assumptions, we applied finite-mixture modelling to estimate a threshold of Ct <21·36 (95% confidence interval 20·29–22·46) to distinguish NV-positive patients for which NV was the likely cause of diarrhoea. We evaluated the validity of the threshold through comparisons with NV antigen ELISA results, and comparisons of Ct values in patients co-infected with rotavirus. We conclude that the use of an appropriate cut-off value in the interpretation of NV real-time RT–PCR results may improve differential diagnosis of enteric infections, and could contribute to improved estimates of the burden of NV disease.

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