Dynamic quantification of avian influenza H7N9(A) virus in a human infection during clinical treatment using droplet digital PCR.

This study involved a human infection with avian influenza H7N9(A) virus in Zhejiang province, the first one after implementing the closure measures of living poultry markets in China. The clinical symptoms, epidemiological and virological characteristics of the case were described briefly, and as the emphasis, H7N9 virus was detected quantitatively and continuously from the collected samples in 10 different periods of the patient's treatment in order to reveal changes of viral load in patient's body during the treatment. This study first used reverse-transcription droplet digital PCR (RT-ddPCR) assays to monitor viral load dynamically for human H7N9 infection, synchronously performing real-time RT-PCR as a reference technology to obtain more comprehensive data for comparison. Our results indicated that RT-ddPCR compared to real-time RT-PCR is more sensitive and accurate for quantifying H7N9 viral load without the use of standard curves. Furthermore it can provide reference data for clinical policies including infectivity judgement, ward transferring and therapy adjustment for the patient during treatment.

[1]  Yuxin Shi,et al.  Emerging H7N9 influenza A (novel reassortant avian-origin) pneumonia: radiologic findings. , 2013, Radiology.

[2]  Chao Wu,et al.  Human infection with a novel avian‐origin influenza A (H7N9) virus: serial chest radiographic and CT findings , 2014, Chinese medical journal.

[3]  Shaghayegh Haghjooy Javanmard,et al.  Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes , 2014, Advanced biomedical research.

[4]  Dalyir I. Pretto,et al.  Screening newborn blood spots for 22q11.2 deletion syndrome using multiplex droplet digital PCR. , 2015, Clinical chemistry.

[5]  Steven Jacobson,et al.  Digital droplet PCR (ddPCR) for the precise quantification of human T-lymphotropic virus 1 proviral loads in peripheral blood and cerebrospinal fluid of HAM/TSP patients and identification of viral mutations , 2014, Journal of NeuroVirology.

[6]  P. Corbisier,et al.  Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction , 2010, Analytical and bioanalytical chemistry.

[7]  H. Doi,et al.  Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys , 2015, PloS one.

[8]  N. Cox,et al.  Global concerns regarding novel influenza A (H7N9) virus infections. , 2013, The New England journal of medicine.

[9]  Don D. Sin,et al.  A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2 , 2014, PloS one.

[10]  Jie Dong,et al.  Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus. , 2018 .

[11]  Sebastien Gallien,et al.  Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR. , 2012, Journal of virological methods.

[12]  Benjamin J. Hindson,et al.  Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification , 2011, Analytical chemistry.

[13]  Elizabeth McCarthy,et al.  Development and Evaluation of a Next-Generation Digital PCR Diagnostic Assay for Ocular Chlamydia trachomatis Infections , 2013, Journal of Clinical Microbiology.

[14]  Kyung-Suk Cho,et al.  Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil , 2014, Applied Microbiology and Biotechnology.

[15]  K. Jerome,et al.  Viral diagnostics in the era of digital polymerase chain reaction. , 2013, Diagnostic microbiology and infectious disease.

[16]  Benjamin J Cowling,et al.  Effect of closure of live poultry markets on poultry-to-person transmission of avian influenza A H7N9 virus: an ecological study , 2014, The Lancet.

[17]  Paul H Dear,et al.  Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine. , 2013, Methods.

[18]  Mojca Milavec,et al.  Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR , 2013, PloS one.

[19]  I. Damon,et al.  Specific qPCR assays for the detection of orf virus, pseudocowpox virus and bovine papular stomatitis virus. , 2013, Journal of virological methods.

[20]  Philippe Corbisier,et al.  Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number , 2009, Analytical and bioanalytical chemistry.

[21]  Christopher M. Hindson,et al.  Absolute quantification by droplet digital PCR versus analog real-time PCR , 2013, Nature Methods.

[22]  Z. Gu,et al.  Comparison of Droplet Digital PCR to Real-Time PCR for Quantitative Detection of Cytomegalovirus , 2012, Journal of Clinical Microbiology.

[23]  C. Foy,et al.  Evaluation of Digital PCR for Absolute RNA Quantification , 2013, PloS one.

[24]  J. Hampe,et al.  Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora , 2004, Journal of Clinical Microbiology.

[25]  Alexandra S. Whale,et al.  Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation , 2012, Nucleic acids research.

[26]  Mingbin Liu,et al.  Surveillance of avian influenza A(H7N9) virus infection in humans and detection of the first imported human case in Taiwan, 3 April to 10 May 2013. , 2013, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[27]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[28]  Ying Wu,et al.  Environmental connections of novel avian-origin H7N9 influenza virus infection and virus adaptation to the human , 2013, Science China Life Sciences.

[29]  S. Vong,et al.  Human infections with avian influenza A(H7N9) virus in China: preliminary assessments of the age and sex distribution. , 2013, Western Pacific surveillance and response journal : WPSAR.

[30]  Maja Ravnikar,et al.  Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples , 2014, Plant Methods.

[31]  Malcolm R Masso,et al.  WORLD HEALTH ORGANIZATION Regional Office for the Western Pacific , 2005 .

[32]  K. Kelsey,et al.  A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood , 2014, Epigenetics.

[33]  Alexandra S. Whale,et al.  Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples , 2013, PloS one.

[34]  P. Deininger,et al.  A droplet digital PCR detection method for rare L1 insertions in tumors , 2014, Mobile DNA.