scholarly journals A Novel Multiplex PCR Based Detection Assay Using Saliva or Nasopharyngeal Samples for SARS-Cov-2, Influenza A and B – Clinical Validation and Utility for Mass Surveillance

2021 ◽  
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Sudha Ananth ◽  
Allan Njau ◽  
Pankaj Ahluwalia ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Influenza A ◽  
Detection Rate ◽  
Limit Of Detection ◽  
Multiplex Assay ◽  
Influenza Viruses ◽  
Clinical Validation ◽  
Rt Pcr ◽  
Specific Assay ◽  
Tract Infections

AbstractBackgroundThe COVID-19 pandemic has resulted in a significant diversion of human and material resources to COVID-19 diagnostics, to the extent that testing of viral pathogens normally contributing to seasonal respiratory tract infections have been markedly neglected. The global health burden due to influenza viruses and co-infection in COVID-19 patients remains undocumented but clearly pose serious public health consequences. To address these clinical and technical challenges, we have optimized and validated a highly sensitive RT-PCR based multiplex assay for the detection of SARS-CoV-2, Influenza A and B viruses in a single test.MethodsThis study evaluated clinical specimens (n=1411) that included 1019 saliva and 392 nasopharyngeal swab (NPS) samples collected in either healthcare or community setting. Samples were tested using two assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp RT-PCR based assay that targets SARS-CoV-2, Influenza viruses A and B. The limit of detection (LoD) studies was conducted as per the FDA guidelines using SARS-CoV-2 and Influenza A and B reference control materials.ResultsOf the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with our multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with our multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. The Ct values for SARS-CoV-2 were comparable between the two assays, whereas the Ct values of the housekeeping gene was significantly lower with multiplex assay compared to SARS-specific assay. The LoD was established as 60 copies/ml for SARS-CoV-2 and 180 copies/ml for Influenza A and B viruses for both saliva and NPS samples.ConclusionThis study presents clinical validation of a multiplex PCR assay for testing SARS-CoV-2, Influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow. This novel assay uses the same instruments, sample types, supplies, and laboratory personnel as needed for the testing of SARS-CoV-2 virus.

scholarly journals A multiplex one-step real-time RT-PCR assay for influenza surveillance

2011 ◽  
Vol 16 (7) ◽  
Author(s):  
I Huber ◽  
H Campe ◽  
D Sebah ◽  
C Hartberger ◽  
R Konrad ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Influenza A ◽  
Multiplex Assay ◽  
Influenza Viruses ◽  
Influenza Surveillance ◽  
Influenza B ◽  
Rt Pcr ◽  
Influenza A H1n1 ◽  
One Step

For surveillance purposes real-time PCR assays for influenza viruses had to be adapted to the pandemic influenza A(H1N1)2009 strain. We combined published primers and probes for influenza A, influenza B and an internal amplification control with a detection system for influenza A(H1N1)2009 to set up a rapid, reliable, simple and cost-effective high-throughput multiplex one-step real-time RT-PCR. The workflow also includes automated sample preparation for high-throughput screening. The lower limit of detection of the multiplex assay was 3.5x102 RNA copies per PCR reaction. The diagnostic sensitivity of the multiplex assay was 87.7%, but increased to 99.4% for influenza-positive samples yielding Ct values of less than 34 cycles in the respective diagnostic assay. High specificity was confirmed by sequencing and correct detection of 15 reference samples from two quality assurance studies. The multiplex PCR was introduced for surveillance of samples from a network of general practitioners and paediatricians in Bavaria, Germany during the influenza pandemic of 2009. Comparison with surveillance data from reported cases proved the reliability of the multiplex assay for influenza surveillance programmes.

scholarly journals Report on Influenza A and B Viruses: Their Coinfection in a Saudi Leukemia Patient

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Fahad N. Almajhdi ◽  
Ghazanfar Ali
Keyword(s):  
Influenza A ◽  
Respiratory Infections ◽  
Clinical Specimen ◽  
Clinical Importance ◽  
Respiratory Illness ◽  
Influenza Viruses ◽  
Clinical Samples ◽  
Rt Pcr ◽  
One Year ◽  
Tract Infections

Purpose. Influenza A and B viruses are the leading cause of respiratory infections in children worldwide, particularly in developing countries. There is a lack of data on coinfection of influenza A and B viruses circulating in Saudi Arabia. In this study, we aimed to identify the circulation of influenza viruses that contribute to respiratory tract infections in Saudi children.Methods. We collected 80 nasopharyngeal aspirates (NPAs) from hospitalized children with acute respiratory illness (ARI) at Riyadh during the period extended from October 2010 till April 2011. Samples were tested for the common respiratory viruses including influenza viruses by RT-PCR.Results. Overall, 6 samples were found positive for influenza A and/or B viruses. Among these positive clinical samples, only one collected sample from a female one-year-old immunocompromised child with leukemia showed a coinfection with influenza A and B viruses. In present study coinfection was confirmed by inoculation of the clinical specimen in specific pathogenfree embryonating chicken eggs and identification of the virus isolates by hemagglutination and one-step RT-PCR.Conclusion. This study opens the scene for studying the role of influenza virus’s coinfection in disease severity and virus evolution. Further studies are required to better understand the clinical importance of viral coinfection.

scholarly journals No evidence of SARS-CoV-2 in hospitalized patients with severe acute respiratory syndrome in five Italian hospitals from 1st November 2019 to 29th February 2020

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260947
Author(s):  
Donatella Panatto ◽  
Andrea Orsi ◽  
Beatrice Marina Pennati ◽  
Piero Luigi Lai ◽  
Stefano Mosca ◽  
...  
Keyword(s):  
Phase I ◽  
Influenza A ◽  
Hospitalized Patients ◽  
Influenza Viruses ◽  
Influenza B ◽  
Molecular Testing ◽  
Rt Pcr ◽  
The Real ◽  
Severe Acute Respiratory Infection ◽  
Novel Coronavirus

Background On 9th January 2020, China CDC reported a novel coronavirus (later named SARS-CoV-2) as the causative agent of the coronavirus disease 2019 (COVID-19). Identifying the first appearance of virus is of epidemiological importance to tracking and mapping the spread of SARS-CoV-2 in a country. We therefore conducted a retrospective observational study to detect SARS-CoV-2 in oropharyngeal samples collected from hospitalized patients with a Severe Acute Respiratory Infection (SARI) enrolled in the DRIVE (Development of Robust and Innovative Vaccine Effectiveness) study in five Italian hospitals (CIRI-IT BIVE hospitals network) (1st November 2019 – 29th February 2020). Objectives To acquire new information on the real trend in SARS-CoV-2 infection during pandemic phase I and to determine the possible early appearance of the virus in Italy. Materials and methods Samples were tested for influenza [RT-PCR assay (A/H1N1, A/H3N2, B/Yam, B/Vic)] in accordance with the DRIVE study protocol. Subsequently, swabs underwent molecular testing for SARS-COV-2. [one-step real-time multiplex retro-transcription (RT) PCR]. Results In the 1683 samples collected, no evidence of SARS-CoV-2 was found. Moreover, 28.3% (477/1683) of swabs were positive for influenza viruses, the majority being type A (358 vs 119 type B). A/H3N2 was predominant among influenza A viruses (55%); among influenza B viruses, B/Victoria was prevalent. The highest influenza incidence rate was reported in patients aged 0–17 years (40.3%) followed by those aged 18–64 years (24.4%) and ≥65 years (14.8%). Conclusions In Italy, some studies have shown the early circulation of SARS-CoV-2 in northern regions, those most severely affected during phase I of the pandemic. In central and southern regions, by contrast no early circulation of the virus was registered. These results are in line with ours. These findings highlight the need to continue to carry out retrospective studies, in order to understand the epidemiology of the novel coronavirus, to better identify the clinical characteristics of COVID-19 in comparison with other acute respiratory illnesses (ARI), and to evaluate the real burden of COVID-19 on the healthcare system.

scholarly journals Methods for Molecular Surveillance of Influenza Used in Macedonia

PRILOZI ◽  
2014 ◽  
Vol 35 (2) ◽  
pp. 25-30
Author(s):  
Golubinka Bosevska ◽  
Elizabeta Janceska ◽  
Gordana Kuzmanovska ◽  
Vladimir Mikik ◽  
Nikola Panovski
Keyword(s):  
Influenza Virus ◽  
Predictive Value ◽  
Influenza A ◽  
Rna Extraction ◽  
Influenza Virus Infection ◽  
Laboratory Diagnosis ◽  
Influenza Viruses ◽  
Rt Pcr ◽  
Two Samples ◽  
Nose And Throat

AbstractThe aim: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country.Materials and methods: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009–2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping.Results: Of 25 samples tested with conventional RT-PCR7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for A/H1pdm and 1(4%) was A/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours.Discussion and conclusion: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.

scholarly journals Detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses based on CRISPR-Cas12a

2020 ◽  
pp. 153537022096379
Author(s):  
Oraphan Mayuramart ◽  
Pattaraporn Nimsamer ◽  
Somruthai Rattanaburi ◽  
Naphat Chantaravisoot ◽  
Kritsada Khongnomnan ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Severe Acute Respiratory Syndrome ◽  
Influenza A ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Influenza Virus A ◽  
B Virus

Due to the common symptoms of COVID-19, patients are similar to influenza-like illness. Therefore, the detection method would be crucial to discriminate between SARS-CoV-2 and influenza virus-infected patients. In this study, CRISPR-Cas12a-based detection was applied for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus which would be a practical and attractive application for screening of patients with COVID-19 and influenza in areas with limited resources. The limit of detection for SARS-CoV-2, influenza A, and influenza B detection was 10, 103, and 103 copies/reaction, respectively. Moreover, the assays yielded no cross-reactivity against other respiratory viruses. The results revealed that the detection of influenza virus and SARS-CoV-2 by using RT-RPA and CRISPR-Cas12a technology reaches 96.23% sensitivity and 100% specificity for SARS-CoV-2 detection. The sensitivity for influenza virus A and B detections was 85.07% and 94.87%, respectively. In addition, the specificity for influenza virus A and B detections was approximately 96%. In conclusion, the RT-RPA with CRISPR-Cas12a assay was an effective method for the screening of influenza viruses and SARS-CoV-2 which could be applied to detect other infectious diseases in the future.

scholarly journals Molecular detection of influenza A(H1N1)pdm09 viruses with M genes from human pandemic strains among Nigerian pigs, 2013–2015: implications and associated risk factors

2017 ◽  
Vol 145 (16) ◽  
pp. 3345-3360 ◽  
Author(s):  
O. A. ADEOLA ◽  
B. O. OLUGASA ◽  
B. O. EMIKPE
Keyword(s):  
Risk Factors ◽  
Influenza A ◽  
Influenza Viruses ◽  
Scan Statistics ◽  
Rt Pcr ◽  
Pandemic Virus ◽  
Influenza A H1n1 ◽  
Pandemic Strains ◽  
Associated Risk Factors ◽  
Ibadan Nigeria

SUMMARYIn the post-pandemic period, influenza A(H1N1)pdm09 virus has been detected in swine populations in different parts of the world. This study was conducted to determine the presence and spatial patterns of this human pandemic virus among Nigerian pigs and identify associated risk factors. Using a two-stage stratified random sampling method, nasal swab specimens were obtained from pigs in Ibadan, Nigeria during the 2013–2014 and 2014–2015 influenza seasons, and the virus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Purified RT-PCR products were sequenced in both directions, and sequences were aligned using MUSCLE. Phylogenetic analysis was conducted in MEGA6. Purely spatial scan statistics and a spatial lag regression model were used to identify spatial clusters and associated risk factors. The virus was detected in both seasons, with an overall prevalence of 8·7%. Phylogenetic analyses revealed that the M genes were similar to those of pandemic strains which circulated in humans prior to and during the study. Cluster analysis revealed a significant primary spatial cluster (RR = 4·71, LLR = 5·66,P= 0·0046), while ‘hours spent with pigs (R2= 0·90,P= 0·0018)’ and ‘hours spent with pigs from different farms (R2= 0·91,P= 0·0001)’ were identified as significant risk factors (P< 0·05). These findings reveal that there is considerable risk of transmission of the pandemic virus, either directly from pig handlers or through fomites, to swine herds in Ibadan, Nigeria. Active circulation of the virus among Nigerian pigs could enhance its reassortment with endemic swine influenza viruses. Campaigns for adoption of biosecurity measures in West African piggeries and abattoirs should be introduced and sustained in order to prevent the emergence of a new influenza epicentre in the sub-region.

scholarly journals Molecular Detection of a Novel Human Influenza (H1N1) of Pandemic Potential by Conventional and Real-Time Quantitative RT-PCR Assays

2009 ◽  
Vol 55 (8) ◽  
pp. 1555-1558 ◽  
Author(s):  
Leo L M Poon ◽  
K H Chan ◽  
G J Smith ◽  
C S W Leung ◽  
Y Guan ◽  
...  
Keyword(s):  
Real Time ◽  
Influenza A ◽  
H1n1 Virus ◽  
Influenza Viruses ◽  
The Novel ◽  
Human Influenza ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Recent Emergence ◽  
Human H5n1

Abstract Background: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed. Methods: We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses. Results: All of the assays had detection limits for the positive control in the range of 1.0 × 10−4 to 2.0 × 10−3 of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses. Conclusions: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.

scholarly journals SalivaAll: Clinical validation of a sensitive test for saliva collected in healthcare and community settings with pooling utility for SARS-CoV-2 mass surveillance.

2020 ◽  
Author(s):  
Nikhil Shri Sahajpal ◽  
Ashis K Mondal ◽  
Sudha Ananth ◽  
Allan Njau ◽  
Pankaj Ahluwalia ◽  
...  
Keyword(s):  
Phase I ◽  
Phase Ii ◽  
Detection Rate ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Community Settings ◽  
Rt Pcr ◽  
Effective Measure ◽  
Pooled Testing ◽  
Mass Surveillance

Background: The adoption of saliva as a specimen type for SARS-CoV-2 mass surveillance can significantly increase population compliance with decreased exposure risk for healthcare workers. However, studies evaluating the clinical performance of saliva compared to nasopharyngeal swab (NPS) samples have demonstrated conflicting results regardless of the collection being in healthcare or community settings. Further, pooled testing with saliva remains a challenge owing to the ambiguous sensitivity, limit of detection (LoD), and processing challenges. To overcome these limitations, SalivaAll protocol was developed and validated as a cost-effective measure that must be used on saliva collected in health care or community settings with pooling utility for SARS-CoV-2 mass surveillance. Methods: The study evaluated 429 matched NPS and saliva samples collected from 344 individuals in either healthcare or community setting. In phase I (protocol U), 240 matched NPS, and saliva samples were tested for SARS-CoV-2 detection by RT-PCR. In phase II (SalivaAll protocol), 189 matched NPS and saliva samples were tested, with an additional sample homogenization step for saliva before RNA extraction, followed by RT-PCR. Eighty-five saliva samples were evaluated with both protocols (U and SalivaAll). Subsequently, adopting SalivaAll protocol, a five-sample pooling strategy was evaluated for saliva samples based on FDA recommendations. Results: In phase I, 28.3% (68/240) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate was lower in saliva compared to NPS samples (50.0% vs. 89.7%). In phase II, 50.2% (95/189) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate for SARS-CoV-2 was higher in saliva compared to NPS testing (97.8% vs. 78.9%). Of the 85 saliva samples evaluated by both protocols, 57.6% (49) tested positive for SARS-CoV-2 with either protocol U, SalivaAll, or both. The detection rate was 100% for samples tested with SalivaAll, whereas it was 36.7% with protocol U. Also, the LoD with SalivaAll protocol was 20 copies/ml. The pooled testing approach demonstrated a 95% positive and 100% negative percent agreement. Conclusion: This single-site study demonstrated the variability of results reported in the literature for saliva samples, and found that the discrepancies are explained by processing challenges associated with saliva samples. We have optimized a protocol for saliva samples that results in higher sensitivity compared to NPS samples and also breaks the barrier to using pooled saliva testing for SARS-CoV-2.

scholarly journals Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study

1999 ◽  
Vol 37 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Britta Gröndahl ◽  
Wolfram Puppe ◽  
Andrea Hoppe ◽  
Inka Kühne ◽  
Josef A. I. Weigl ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Reverse Transcription ◽  
Influenza A ◽  
Respiratory Tract Infections ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Tract Infections ◽  
Type 3

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus,Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.

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