Difference between revisions of "Influenza A"

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[[File:Influenzastructure.png|thumb|360px|Structure of the influenza virus.<ref name="Arti1">Host Protective Immune Responses against Influenza A Virus Infection, MDPI, 2020, Hi Eun Jung, Heung Kyu Lee, https://www.mdpi.com/1999-4915/12/5/504/htm</ref>]]
  
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== General Information==
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== General information==
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The theme of SensUs 2021 is acute respiratory viruses. The current Covid-19 pandemic has made it apparent that large virus outbreaks can cause immense harm to human health and can disrupt society as a whole. The most common respiratory virus is influenza A. Therefore, the influenza virus serves as an interesting biomarker for this year’s Competition.
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The theme of SensUs 2021 is acute respiratory viruses. The current Covid-19 pandemic has made it apparent that large virus outbreaks can cause immense harm to human health and can disrupt society as a whole. One of the most common respiratory viruses is influenza A. Therefore, the influenza virus serves as an interesting research topic for this year’s Competition.
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The influenza A virus is known to cause influenza in birds and some mammals, like humans. Several subtypes of the influenza A virus have been isolated from wild birds. Occasionally, viruses can be transmitted from wild birds to domestic animals, like chickens or pigs. This may give rise to human influenza.<ref name="Arti2">Transmission of Avian Influenza A Viruses Between Animals and People, CDC, 2015, https://www.cdc.gov/flu/avianflu/virus-transmission.htm</ref>
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Influenza A viruses are negative-sense, single-stranded RNA viruses. Subtypes of influenza A are characterized by proteins on the outermost membrane of the virus, called hemagglutinin (H or HA) and neuraminidase (N or NA). H and N are the antigens of the virus, and play an important role in the interaction between the host’s immunological response and the virus. Recently, researchers have reported the discovery of an antibody that is generally effective against all subtypes of the influenza A virus.<ref name="Arti3">Super antibody' fights off flu, BBC, 2011, James Gallagher, https://www.bbc.com/news/health-14324901</ref> Hemagglutinin will serve as this year's biomarker. In infected patients, bound hemagglutinin indicates the presence of the influenza virus. In the Testing Event, unbound hemagglutinin proteins will be used to mimic the immunologic response of the virus.
  
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The influenza A virus is known to cause influenza in birds and some mammals, like humans. Different subtypes of the influenza A virus have been isolated from wild birds. Some subtypes of the influenza A virus can cause severe symptoms both in domestic poultry and (rarely) in humans and can even lead to death. Occasionally, viruses can be transmitted from wild birds to domestic animals, like chickens or pigs. This may give rise to human influenza.
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The subtype which will be used in SensUs 2021 is H1N1. Historically, H1N1 has been responsible for most deaths due to influenza. It is a popular influenza strain for research purposes.<ref name="Arti4">Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection, Research Gate, 2020, Sun, Honglei & Xiao, Yihong & Liu, Jiyu & Wang, Dayan & Li, Fangtao & Wang, Chenxi & Li, Chong & Zhu, Junda & Song, Jingwei & Sun, Haoran & Zhimin, Jiang & Liu, Litao & Zhang, Xin & Wei, Kai & Dongjun, Hou & Pu, Juan & Sun, Yipeng & Tong, Qi & Bi, Yuhai & Liu, Jinhua https://www.researchgate.net/publication/342555087_Prevalent_Eurasian_avian-like_H1N1_swine_influenza_virus_with_2009_pandemic_viral_genes_facilitating_human_infection</ref><ref name="Arti5">Comparison of Hospitalized Patients With ARDS Caused by COVID-19 and H1N1, CHEST, Xiao Tang, Rong-Hui Du, Rui Wang, Tan-Ze Cao, Lu-Lu Guan, Cheng-Qing Yang, Qi Zhu, Ming Hu, Xu-Yan Li, Ying Li, Li-Rong Liang, Zhao-Hui Tong, Bing Sun, Peng Peng, Huan-Zhong Shi, 2020, https://www.sciencedirect.com/science/article/pii/S0012369220305584</ref><ref name="Arti6">Landscape of coordinated immune responses to H1N1 challenge in humans, Journal of Clinical Investigation, 2020, Zainab Rahil, Rebecca Leylek, Christian M. Schürch, Han Chen, Zach Bjornson-Hooper, Shannon R. Christensen, Pier Federico Gherardini, Salil S. Bhate, Matthew H. Spitzer, Gabriela K. Fragiadakis, Nilanjan Mukherjee, Nelson Kim, Sizun Jiang, Jennifer Yo, Brice Gaudilliere, Melton Affrime, Bonnie Bock, Scott E. Hensley, Juliana Idoyaga, Nima Aghaeepour, Kenneth Kim, Garry P. Nolan, David R. McIlwain https://www.sciencedirect.com/science/article/pii/S0012369220305584</ref> Due to its popularity among researchers, antigens and antibodies are commercially available, making H1N1 suitable as a target for the SensUs Competition.
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Influenza A viruses are negative-sense, single-stranded RNA viruses. Different subtypes of influenza A exist, these are characterized by proteins on the outermost membrane of the virus, called hemagglutinin (H or HA) and neuraminidase (N or NA). H and N are the antigens of the virus, and play an important role in the interaction between the host’s immunological response and the virus. Recently, researchers have reported the discovery of an antibody which is generally effective against all types of the influenza A virus.
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Influenza A vaccines for humans have been developed. New versions of the vaccines are developed twice per year for use all over the world, which is necessary due to rapid mutations of the influenza virus. Every year during flu season, a large part of the population is vaccinated in order to protect individuals against the virus. However, due to unforeseen mutations of the virus, it might be possible that in a certain year a vaccine will prove ineffective. In that case large portions of the population would be at risk and a pandemic could occur. The probability of a major influenza A pandemic is estimated to be around 0.5-1% each year.<ref name="Arti7">Pandemic risk: how large are the expected losses?, WHO, 2017, Victoria Y Fan, Dean T Jamisonb & Lawrence H Summers, https://www.who.int/bulletin/volumes/96/2/17-199588.pdf</ref>
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The subtype which will be used in SensUs 2021 is H1N1. Historically, H1N1 has been responsible for most deaths due to influenza. It is a popular influenza strain for research purposes [link, link, link] . Due to its popularity among researchers, antigens and antibodies are commercially available, making H1N1 suitable as a target for the SensUs competition.
 
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Influenza A vaccines for humans have been developed. New versions of the vaccines are developed twice per year for use all over the world, which is necessary due to rapid mutations of the influenza virus. Every year during flu season, a large part of the population is vaccinated in order to protect individuals against the virus. However, due to unforeseen mutations of the virus, it might be possible that in a certain year a vaccine will prove ineffective. In that case large portions of the population would be at risk and a pandemic could occur. The probability of a major influenza A pandemic is estimated to be around 0.5-1% each year.
 
  
 
==History of influenza (A)==
 
==History of influenza (A)==
 
   
 
   
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A lack of data up until 1500 AC complicates the research on influenza before that period.[206] Possibly the first influenza pandemic occurred around 6000 BC in China. The symptoms of human influenza seem to have been clearly described by Hippocrates, roughly 2,400 years ago[207][208]. Although the virus seems to have caused epidemics throughout human history, historical data on influenza is difficult to interpret, due to the fact that symptoms of influenza are similar to those found in other respiratory diseases, like RSV (respiratory syncytial virus).
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A lack of data up until 1500 AC complicates the research on influenza before that period.<ref name="Arti8">Internet‐Based Intelligence in Public Health Emergencies, NATO Science for Peace and Security Series - E: Human and Societal Dynamics, 2013 Mordini E., Green M., https://www.iospress.nl/book/internet%E2%80%90based-intelligence-in-public-health-emergencies/</ref> Possibly the first influenza pandemic occurred around 6000 BC in China. The symptoms of human influenza seem to have been clearly described by Hippocrates, roughly 2,400 years ago<ref name="Arti9">2,500-year Evolution of the Term Epidemic, Emerging infectious diseases, 2006, Martin PM, Martin-Granel E, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373038/</ref> Although the virus seems to have caused epidemics throughout human history, historical data on influenza is difficult to interpret, due to the fact that symptoms of influenza are similar to those found in other respiratory diseases, like respiratory syncytial virus (RSV).
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The most infamous and lethal outbreak was the 1918 flu pandemic (Spanish flu) (type A influenza, H1N1 subtype), which lasted into 1920. The number of deaths is unknown, but estimates range from 17 to 100 million people. This pandemic has been described as "the greatest medical holocaust in history" and may have killed as many people as the plague (Black Death). This huge death toll was caused by an extremely high infection rate of up to 50% and the severity of the symptoms, suspected to be caused by cytokine storms in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules called cytokines.
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One of the most recent outbreaks of influenza was the 2009 Swine Flu. Similar to the Spanish Flu, it was also of the subtype H1N1. The death toll of the 2009 pandemic is estimated to be around 150,000 to 575,000.
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The most infamous and lethal outbreak was the 1918 flu pandemic, which lasted until 1920. The number of deaths is unknown, but estimates range from 17 to 100 million people.<ref name="Arti10">Reassessing the Global Mortality Burden of the 1918 Influenza Pandemic, American journal of epidemiology, 2018, Spreeuwenberg P, Kroneman M, Paget J, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314216/</ref>. This pandemic has been described as "the greatest medical holocaust in history"<ref name="Arti11"> Reviewing the History of Pandemic Influenza: Understanding Patterns of Emergence and Transmission
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, Pathogens, 2016, Saunders-Hastings PR, Krewski D, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198166/</ref> and may have killed as many people as the plague (Black Death). This huge death toll was caused by an extremely high infection rate of up to 50% and the severity of the symptoms, suspected to be caused by cytokine storms in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules called cytokines.
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One of the most recent outbreaks of influenza was the 2009 Swine Flu. Similar to the Spanish Flu, it was also of the subtype H1N1. The death toll of the 2009 pandemic is estimated to be around 150,000 to 575,000.<ref name="Arti12">2009 H1N1 Pandemic (H1N1pdm09 virus), centers for disease control and prevention, 2010, https://www.cdc.gov/flu/pandemic-resources/2009-h1n1-pandemic.html</ref>
  
 
==Structure of the influenza virus==
 
==Structure of the influenza virus==
 
   
 
   
 
   
 
   
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The virus particle (virion) is 80–120 nanometers in diameter. The virion shape can be spherical, elliptical, or even filamentous with a length of tens of micrometers. The virion is made up of a viral envelope containing two main types of proteins, wrapped around a central core.
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The virus particle (virion) is 80–120 nanometers in diameter.<ref name="Arti13">Native Morphology of Influenza Virions, Frontiers in microbiology, 2011, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249889/</ref> The virion shape can be spherical, elliptical, or even filamentous with a length of tens of micrometers<ref name="Arti13">Native Morphology of Influenza Virions, Frontiers in microbiology, 2011, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249889/</ref>. The virion is made up of a viral envelope containing two main types of proteins, wrapped around a central core. The two large proteins found on the outside of viral particles are hemagglutinin (HA) and neuraminidase (NA). HA is a protein that mediates binding of the virion to target cells and entry of the viral genome into the target cell, and therefore plays an important role in infecting healthy cells. NA is involved in releasing the progeny viruses once a cell has been infected and has started producing the virus itself.<ref name="Arti14">Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase, Virology journal , 2013, Cohen M, Zhang XQ, Senaati HP, Chen HW, Varki NM, Schooley RT, Gagneux P, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842836/</ref>
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The two large proteins found on the outside of viral particles are hemagglutinin (HA) and neuraminidase (NA). HA is a protein that mediates binding of the virion to target cells and entry of the viral genome into the target cell, and therefore plays an important role in infecting healthy cells. NA is involved in releasing the progeny viruses once a cell has been infected and has started producing the virus itself.
 
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These two proteins are a target of interest for antiviral drugs. Furthermore, they are also the antigen proteins to which a host antibodies can bind and trigger an immune response. Influenza type A viruses are categorized into different subtypes, or strains, based on which type of these two proteins is present on the surface of the virion. Currently, there are 16 subtypes of HA and 9 subtypes of NA known to exist. The most prevalent form of the different subtypes is H1N1. Single hemagglutinin-neuraminidase proteins, in which both HA and NA are found in a single protein, also exist. However, these will not be used in SensUs 2021.
 
  
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===Valproate Mode of Action===
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These two proteins are a target of interest for antiviral drugs.<ref name="Arti15">Recent Strategies in the Search for New Anti-Influenza Therapies, Current Drug Targets , 2003, J.C. Wilson, M. von Itzstein, https://www.eurekaselect.com/63785/article</ref> Furthermore, they are also the antigen proteins to which a host antibodies can bind and trigger an immune response. Influenza type A viruses are categorized into different subtypes, or strains, based on which type of these two proteins is present on the surface of the virion. Currently, there are 16 subtypes of HA and 9 subtypes of NA known to exist. The most prevalent form of the different subtypes is H1N1. Single hemagglutinin-neuraminidase proteins, in which both HA and NA are found in a single protein, also exist. However, these will not be used in SensUs 2021.
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The exact mode of action of valproate on molecular level is unknown, as there are many different substances that participate simultaneously in the regulation of neuronal activity. <ref name=”[15]”> Sodium valproate (Epilim, Epival, Episenta). (2018, June 5) , at [https://www.netdoctor.co.uk/medicines/brain-nervous-system/a6665/epilim-sodium-valproate/ “https://www.netdoctor.co.uk/medicines/brain-nervous-system/a6665/epilim-sodium-valproate/”].</ref>  VPA is connected to cortical inhibition in order to contribute to ‘neural synchrony’ and is known to provide protection from neural degradation and damage. <ref name=”[16]”> Williams, R. S. B., Cheng, L., Mudge, A. W., & Harwood, A. J. (2002). A common mechanism of action for three mood-stabilizing drugs , at [https://www.nature.com/articles/417292a “https://www.nature.com/articles/417292a”].</ref>
 
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VPA inhibits histone deacetylase, which causes hyperacetylation of histones. The drug is furthermore connected to inositol depletion by preventing the gene prolyl oligopeptidase to be expressed through indirect inhibition of myo-inositoal-1-phophate-synthetase. <ref name=”[16]”> Williams, R. S. B., Cheng, L., Mudge, A. W., & Harwood, A. J. (2002). A common mechanism of action for three mood-stabilizing drugs , at [https://www.nature.com/articles/417292a “https://www.nature.com/articles/417292a”].</ref> In terms of epilepsy, it is believed that VPA is affecting the activity of GABA. By increasing the activity of GABA, the epileptic attacks are prevented. This is established through inhibition of succinic semialdehyde dehydrogenase which then increases the amount of succinic semialdehyde and subsequently increases GABA neurotransmission. <ref name=”[37]”> Flyyn, S., & Babi, M. A. (2017). Pathophysiology of Epilepsy , at [https://www.sciencedirect.com/topics/medicine-and-dentistry/pathophysiology-of-epilepsy “https://www.sciencedirect.com/topics/medicine-and-dentistry/pathophysiology-of-epilepsy”].</ref>
 
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VPA affects the extracellular signal-regulated kinase pathway or ERK (Extracellular Receptor Kinase), which results in phosphorylation of ERK 1/2 . This has an effect on the expression of proteins that contribute to the plasticity of neurons and neuronal growth. The downside of this effect of VPA is the additional increase in GABA activity. VPA has an influence on fatty acids production, leading to lower membrane fluidity due to the presence of less sterols and glycerolipids. Subsequently, this increases the action potential threshold of the membrane and contributes to the antiepileptic effects of valproate . <ref name=”[17]”> , . Drugbank, Valproic Acid Retrieved from November 25, 2019, at [https://www.drugbank.ca/drugs/DB00313
 
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“https://www.drugbank.ca/drugs/DB00313”].</ref>
 
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===Pharmacokinetics of VPA===
 
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When introduced orally, VPA is found to be absorbed in a period of 4 hours. However, introducing it in other ways results in the same Cmax, AUC and Cmin in a steady state, while all the differences in absorption are neglected. When VPA is taken as extended release tablet and combined with meals, the absorption time increases from 4 to 8 hours. On the other hand, the increase in absorption time in the same situation increases from 3.3 to 4.8 hours when taken as a sprinkle tablet. There is 90% bioavailability of VPA in all oral forms. On the other hand, there is 100% bioavailability in enteric-coated forms.
 
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<ref name=”[18]”> Gugler, R., & Unruh, G. E. von. (2012, December 13). Clinical Pharmacokinetics of Valproic Acid , at [https://link.springer.com/article/10.2165/00003088-198005010-00002 “https://link.springer.com/article/10.2165/00003088-198005010-00002”].</ref>The average half-life of VPA is 13-19 hours, the volume of distribution is 11 L/1.73 m2  . The protein binding is linear at low concentrations, but non-linear and decreased at high concentrations, which may be due to the different affinity binding sites of albumin. Parts of the drug can be metabolized in different ways. The most relevant metabolism of VPA would be glucuronide conjugates and mitochodrial-ß oxidation. <ref name=”[17]”> , . Drugbank, Valproic Acid Retrieved from November 25, 2019, at [https://www.drugbank.ca/drugs/DB00313
 
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“https://www.drugbank.ca/drugs/DB00313”].</ref>
 
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Only 3% of the drug is eliminated through urine, about 30-50% is eliminated through hepatic metabolism, and about 40% being excreted through mitochondrial-beta oxidation. <ref name=”[18]”> Gugler, R., & Unruh, G. E. von. (2012, December 13). Clinical Pharmacokinetics of Valproic Acid , at [https://link.springer.com/article/10.2165/00003088-198005010-00002 “https://link.springer.com/article/10.2165/00003088-198005010-00002”].</ref>
 
  
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===Efficacy and influence on hepatic function===
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==Mechanism of infection==
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Optimal dose and effects are achieved with upper limit of 60 mg/kg/day.  If the desired effects are not achieved (in other words, the seizures are not gone or the side effects are too strong), testing for blood levels of VPA needs to be executed in order to determine whether they fall within the optimal range of total VPA 50-100 µg/mL. Otherwise, the dose is altered in agreement with the doctor according to the individual state of the patient, his/her other conditions or other medication that they take. The toxicity level of valproate in blood, although not very conclusive, is taken as 150 µg/mL<ref name=”[19]”> valproic Acid Dosage Guide with Precautions. (2019, March 28), at [https://www.drugs.com/dosage/valproic-acid.html “https://www.drugs.com/dosage/valproic-acid.html”].</ref>
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[[File:Flu-infect.jpg |right|thumb|200px|An influenza virus infecting a cell.<ref name="Arti31">Sugars on Cell Surface Are Key to Flu Infections, National Institute of Health, 2008, https://www.nih.gov/news-events/nih-research-matters/sugars-cell-surface-are-key-flu-infections</ref>]]
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VPA impacts the hepatic drug metabolism by inhibiting it and displacing other strongly bound drugs from proteins, which implies that dosage changes need to be considered when using it in combination with other drugs. <ref name=”[20]”> Valproic Acid (2018, October 1)  , at [https://labtestsonline.org/tests/valproic-acid“https://labtestsonline.org/tests/valproic-acid”].</ref> The normal limits of valproate in blood of 70-100 kg person is 1000-3000 mg/day  <ref name=”[46]”> Farmacotherapeutisch Kompas. Retrieved December 19, 2019, at [https://www.farmacotherapeutischkompas.nl/bladeren/preparaatteksten/v/valproinezuur“https://www.farmacotherapeutischkompas.nl/bladeren/preparaatteksten/v/valproinezuur”].</ref>
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The involvement of the hemagglutinin and neuraminidase proteins in the infection of H1N1 is essential for the virus reproduction, and a more specific mechanism is discussed below.
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The hemagglutinin protein (HA) has the role of searching for the sialic acid receptors in respiratory-lining cell membranes. Upon binding of this protein and the receptor, fusion of the virus and the cell membrane is facilitated with the help of glycan proteins. The virus then enters the cell where it sheds its shell and approaches the cell’s nucleus. Using the host replication mechanisms, copies of the virus are made. At this step of the infection process, important viral proteins are synthesized. These newly replicated viral elements subsequently leave through the cell membrane and infect other cells. To inhibit the exit of the viral components, sialic acid receptors on the cell can bind the HA glycoproteins. This is where viral evolution/mutability can play a role in the expansion of the capabilities of the virus.
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The neuraminidase glycoprotein (N) has the role of cleaving the sialic acid receptors, allowing the exit of the viral components which then go in search of a new host.
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After infection is complete, the H1N1 virus triggers cell apoptosis, leading to the death of the cell and spread of the virions.
  
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==Number of patients==
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==Medical application==
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Epilepsy affects around 50 million people worldwide. This includes people having less than one seizure per year. The prevalence of active epilepsy (i.e. patients who have frequent seizures or use medication) is between 0.4% and 1%. On a global scale, an estimated five million people are diagnosed with epilepsy each year.
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The influenza virus spreads when people with the flu cough, sneeze or talk. Infected people transfer tiny droplets to people close to them and infect them by the handover of droplets. During an outbreak, it is important to be able to control and prevent the virus from spreading further, for example, by implementing control measures. Those control measures can be determined based on the reproduction number. The reproduction number is defined as: “the expected number of secondary cases produced by a single infection in a completely susceptible population”.<ref name="Arti16">Notes on R0, 2007, James Holland Jones, https://web.stanford.edu/~jhj1/teachingdocs/Jones-on-R0.pdfe</ref> A clear overview of the number of infected people is needed to calculate the reproduction number.
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Epileptic seizures can be controlled by using anti-epileptic drugs (AEDs). About 70% of epileptic patients becomes free of seizures by appropriate AEDs. Low-cost treatments are available, with daily medication that costs as little as US$ 5 per year.<ref name=”[21]”> WHO. (2019, June 20). Epilepsy. Retrieved October 30, 2019 , at [https://www.who.int/news-room/fact-sheets/detail/epilepsy “https://www.who.int/news-room/fact-sheets/detail/epilepsy”].</ref>
 
  
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==Medical use and TDM==
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For disease control, it is crucial to know the infection rate in a specific region. The results may influence critical decisions such as whether to perform other diagnostic testing or to implement infection prevention and control measures for influenza. Furthermore, manpower can be in short supply during a pandemic and the speed at which someone can be tested and receive the result is of vital importance.
  
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Valproate (VPA) is usually taken as tablets of 250 mg or as a syrup with 250 mg per ml, but this may vary per product <ref name=”[39]”> Valproic Acid Capsules - FDA prescribing information, side effects and uses. (2018, June 1).Retrieved November 26, 2019, at [https://www.drugs.com/pro/valproic-acid-capsules.html“https://www.drugs.com/pro/valproic-acid-capsules.html”].</ref>.The dose of VPA is at first taken in small amounts and is then gradually increased until the satisfactory dose is reached.
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Biosensors that will be developed in SensUs 2021 are envisioned to be used outside the hospital in a point-of-care (POC) setting. Inside the hospital there is not a big advantage, as there are already very specific and accurate tests available for that setting.<ref name="Arti28">Diagnosing Flu, CDC, 2020, https://www.cdc.gov/flu/symptoms/testing.htm</ref><ref name="Arti29">Information on Rapid Molecular Assays, RT-PCR, and other Molecular Assays for Diagnosis of Influenza Virus Infection, CDC, 2020, https://www.cdc.gov/flu/professionals/diagnosis/molecular-assays.htm</ref> Important applications are fast testing at the general practioner and at home. Therefore the test should be easy to use. The biosensors will be designed to enable a fast yes/no answer, based on measuring the concentration of hemagglutinin particles in the sample. The biosensors will not distinguish between different virus subtypes, as the subtype causing the pandemic is assumed to be known.
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Testing for valproate levels is important to check if the blood levels are within normal therapeutic range <ref name=”[20]”> Valproic Acid (2018, October 1)  , at [https://labtestsonline.org/tests/valproic-acid“https://labtestsonline.org/tests/valproic-acid”].</ref>
 
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. The test should measure  the free concentration (i.e. the concentration of unbound valproate) as this fraction is pharmacologically active.
 
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The recommended levels of unbound valproate are 6-22 µg/mL in blood <ref name=”[20]”> Valproic Acid (2018, October 1)  , at [https://labtestsonline.org/tests/valproic-acid“https://labtestsonline.org/tests/valproic-acid”].</ref>. For epilepsy patients, the range for the treatment of total valproate should be 50-100 µg/mL. Overall, if a patient has a VPA concentration that falls within this range, does not have recurrent seizures and has minimal side effects, then the dose is said to be suitable<ref name=”[20]”> Valproic Acid (2018, October 1)  , at [https://labtestsonline.org/tests/valproic-acid“https://labtestsonline.org/tests/valproic-acid”].</ref>
 
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. To provide a patient their right amount of medication, therapeutic drug monitoring (TDM) is implemented in hospitals, because every patient responds differently to a certain dose of medication.
 
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VPA is a conventional drug that is used as first line monotherapy for idiopathic generalized epilepsies. Its effectiveness is not clearly conclusive and varies between different patients. The doses are not generalized and depend on patient’s age and weight. Doses are taken every day, with some patients even two times a day. It is not advised, but sometimes necessary, to take VPA in combination with other seizure drugs (e.g., ethosuximide, lamotrigine, phenytoin, rufinamide, topira mate), some antidepressants or certain antibiotics<ref name=”[20]”> Valproic Acid (2018, October 1)  , at [https://labtestsonline.org/tests/valproic-acid“https://labtestsonline.org/tests/valproic-acid”].</ref>. Medications based on VPA are harmful for the unborn child. If valproate is taken during pregnancy, research has shown that up to 4 in 10 babies are at risk of developmental disorders, and approximately 1 in 10 are at risk of birth defects. <ref name=”[22]”>  gov.uk. (2018, March 23), at [https://www.gov.uk/guidance/valproate-use-by-women-and-girls“https://www.gov.uk/guidance/valproate-use-by-women-and-girls”].</ref>
 
  
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==Safety & Lab protocols==
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==State of the Art==
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===Safety===
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There are various influenza tests available on the market. The most common tests are ‘rapid influenza diagnostic tests’, also called RIDTs. RIDTs provide results in a qualitative way within approximately 10-15 minutes and work by detecting the parts of the virus that stimulate an immune response.<ref name="Arti17">Rapid Influenza Diagnostic Tests, 2016, Centers for Disease Control and Prevention, https://www.cdc.gov/flu/professionals/diagnosis/clinician_guidance_ridt.htm</ref>
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All anti-epileptic drugs have side-effects, including VPA. The risk of hyperammonemia is nearly 40% in patients ingesting intravenous VPA in the ICU setting <ref name=”[4]”>  Lind, J., & Nordlund, P. (2019, July). Intravenous use of valproic acid in status epilepticus is associated with high risk of hyperammonemia, Seizure Vol. 69. Retrieved October 23, 2019, at [https://doi.org/10.1016/j.seizure.2019.03.020“https://doi.org/10.1016/j.seizure.2019.03.020”].</ref>
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The table contains examples of available rapid influenza diagnostic tests that provide results in 10-15 minutes. In the SensUs Competition, a saliva based test will be developed. Moreover, SensUs strives to stimulate developement of sensors that provide results within 5-10 minutes and that are as sensitive as possible. The SensUs Competition aims to innovate the field of influenza biosensing by using saliva as a matrix, by improving the speed of the test, and by targeting a high sensitivity.
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. Other adverse effects include thrombocytopenia and endocrine effects of women. Valproate is associated with a dose-related teratogenicity rate, with risk of major malformation higher than 30% at doses greater than 1100 mg/d. In utero exposure is also linked to dose-dependent reduced verbal IQ and autism<ref name=”[6]”> Abou-Khalil, & Bassel, W. (2019). Update on Antiepileptic Drugs 2019. Retrieved November 6, 2019, at [ https://insights.ovid.com/crossref?an=00132979-201904000-00014“ https://insights.ovid.com/crossref?an=00132979-201904000-00014”].</ref>
 
−
.
 
−
Children, who were exposed to VPA during birth, have a possible chance of a major congenital malformation (MCM) <ref name=”[7]”>  Morrow , J., Russell, A., Guthrie, E., Parsons, L., Robertson, I., Waddell, R., … Craig, J. (2006, January 17). Malformation risks of antiepileptic drugs in pregnancy: a prospective study from the UK Epilepsy and Pregnancy Register. Retrieved October 23, 2019, at [https://jnnp.bmj.com/content/77/2/193“https://jnnp.bmj.com/content/77/2/193”].</ref>
 
−
. The risk of having severe consequences for these infants exposed to sodium valproate in utero has been estimated between 6% and 12%<ref name=”[8]”>  Use of Sodium Valproate in Pregnancy. (2014, December 15). Retrieved October 23, 2019, at [https://www.medsafe.govt.nz/profs/PUArticles/December2014SodiumValproate.htm“https://www.medsafe.govt.nz/profs/PUArticles/December2014SodiumValproate.htm”].</ref>
 
−
. This can be prevented by reducing the dose of the drug. An example of endocrine effects might be idiosyncratic liver toxicity <ref name=”[9]”> Stewart, J. D., Horvath, R., Baruffini, E., Ferrero, I., Bulst, S., Watkins, P. B., … Chinnery, P. F. (2010, November). POLG determines the risk of sodium valproate induced liver toxicity. Retrieved October 23, 2019, at [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841971/“https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841971/”].</ref>
 
  
−
===Lab Protocols===
+
{| class="wikitable" style="margin-bottom:0"
 +
!Manufacturer
 +
!Product
 +
!CLIA Waived
 +
!Platform
 +
!Sensitivity
 +
(PPA*)
 +
!Specificity
 +
(NPA**)
 +
!Sample type
 +
!LOD*** (H1N1)
 +
TCID50/mL****
 +
|-
 +
|Abbott<ref name="Arti18">Abbott RIDT Product Information, Abbott, https://www.globalpointofcare.abbott/nl/product-details/binaxnow-influenza-a-and-b.html</ref>
 +
|Binax Now Influenza A & B Card 2
 +
|Yes
 +
|DIGIVAL
 +
|70-89%
 +
|90-99%
 +
|NPS, NS direct
 +
|N/A
 +
|-
 +
|Becton Dickinson & Co.<ref name="Arti19">Becton Dickinson & Co. RIDT Product Information, Becton Dickinson & Co., https://www.bd.com/en-us/offerings/capabilities/microbiology-solutions/point-of-care-testing/veritor-system</ref>
 +
|BD Veritor™ Flu A + B
 +
|Yes
 +
|BD veritor Reader
 +
|82%
 +
|98%
 +
|NPS, NS direct
 +
|3.3*10<sup>2</sup>-5.0*10<sup>3</sup>
 +
|-
 +
|Becton Dickinson & Co.<ref name="Arti19">Becton Dickinson & Co. RIDT Product Information, Becton Dickinson & Co., https://www.bd.com/en-us/offerings/capabilities/microbiology-solutions/point-of-care-testing/veritor-system</ref>
 +
|BD Veritor™ Flu A + B
 +
|Yes
 +
|BD veritor plus Analyzer
 +
|83,6%
 +
|97,5%
 +
|NPS, NS direct
 +
|3.3*10<sup>2</sup>-5.0*10<sup>3</sup>
 +
|-
 +
|Quidel Corp.<ref name="Arti20">Quidel RIDT Product Information, Quidel, https://www.quidel.com/immunoassays/rapid-influenza-tests/sofia-influenza-fia</ref>
 +
|Sofia® Influenza A + B FIA
 +
|Yes
 +
|Sofia(2) FIA Analyzer
 +
|90-99%
 +
|95-96%
 +
|NS, NPS, NPA, NPW direct, NP, NPA, NPW in VTM
 +
|N/A
 +
|-
 +
|Quidel Corp<ref name="Arti21">Quidel QuickVue RIDT Product Information, Quidel, https://www.quidel.com/sites/default/files/product/documents/EF1350313EN00_1.pdf</ref>
 +
|QuickVue® Influenza A + B
 +
|Yes
 +
|N/A
 +
|81,5%
 +
|97,8%
 +
|NS, NPS, NPA, NPW direct, NP, NPA, NPW in VTM
 +
|1.63*10<sup>3</sup>-4.4*10<sup>3</sup>
 +
|-
 +
|Princeton BioMeditech Corp.<ref name="Arti22">BioSing RIDT Product Information, Princeton BioMeditech Corp., http://punchout.medline.com/product/BioSign-Rapid-Flu-AB-Antigen-Panel-Test-by-Princeton-BioMeditech/Influenza-Testing/Z05-PF176440?question=&index=P11&indexCount=11#mrkSpec</ref>
 +
|BioSign® Flu A & B
 +
LABSCO
 +
|Yes
 +
|N/A
 +
|89,2%
 +
|99,4%
 +
|NS, NPS direct, NPA(waived), NPW(not waived)
 +
|N/A
 +
|-
 +
|Remel/Thermo Fisher.<ref name="Arti23">XPECT™ RIDT Product Information, Remel/Thermo Fisher, https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FMBD%2FInstructions%2FIFU24600%2520.pdf&title=WHBlY3QgRmx1IEEgYW5kIEI=</ref>
 +
|XPECT™ Flu A & B
 +
LABSCO
 +
|No
 +
|N/A
 +
|89-100%
 +
|100%
 +
|NW, NS
 +
|1.63*10<sup>3</sup>-4.41*10<sup>3</sup>
 +
|-
 +
|Sekisui Diagnostics, <ref name="Arti24">Acucy Influenza A&B Test Product Information, Sekisui Diagnostics, https://www.sekisuidiagnostics.com/wp-content/uploads/2019/08/Acucy-Influenza-AB-Package-Insert.pdf</ref>
 +
|Acucy Influenza A&B Test
 +
|Yes
 +
|The Acucy System
 +
|96,4%
 +
|96,0%
 +
|NPS, NS direct
 +
|1.4*10<sup>1</sup>
 +
|-
 +
|Sekisui Diagnostics, <ref name="Arti25">OSOM Ultra Plus Flu A&B Test Product Information, Sekisui Diagnostics, https://www.sekisuidiagnostics.com/wp-content/uploads/2019/04/OSOM_P-52631-D__web.pdf</ref>
 +
|OSOM Ultra Plus Flu A&B Test
 +
|Yes
 +
|N/A
 +
|90,2-92,2%
 +
|75,1-85,7%
 +
|NPS, NPA, NPW, NS
 +
|1.05*10<sup>2</sup>
 +
|-
 +
|Access Bio, Inc., <ref name="Arti26">CareStart Flu A&B Plus Product Information, Access Bio, Inc., https://www.accessdata.fda.gov/cdrh_docs/pdf19/K191514.pdf</ref>
 +
|CareStart Flu A&B Plus
 +
|No
 +
|N/A
 +
|79,9%
 +
|98,4%
 +
|NPS
 +
|5.0*10<sup>3</sup>-9.6*10<sup>3</sup>
  
−
VPA is considered thermodynamically stable, which indicates that it is not reactive under normal environmental conditions. It should be stored in a metal can or drum and kept away from incompatible materials such as oxidizing agents, bases and strong reducing agents as ignition may result.
 
  
−
Working with VPA can be irritating when inhaled or when getting directly in contact with the yes. To prevent this, VPA needs to be handled in a fume hood, safety goggles, lab coats, and gloves need to worn. Direct skin contact or ingestion of VPA should be avoided. In case of contact, flush the specific body part and go to the doctor without delay <ref name=”[10]”> Chemwatch: 15242 Version No: 7.1.1.1 Safety Data Sheet (Conforms to Regulation (EU) No 2015/830)</ref>
+
|}<div style="margin-bottom:1em"><sub>''Table 1: Different rapid diagnostic tests for infuenza A''<br />
 +
'''*'''PPA: positive percent agreement (True positive / total true positive)
  
−
==State of the art==
+
'''**'''NPA: negative percent agreement (True negative / total true negative)
  
 +
'''***'''LOD: Limit of detection
 +
'''****'''TCID50/mL represents the viral load at which 50% of cells are infected when a solution containing the virus is added to the cell culture.<ref name="Arti32">Limits of detection for FDA-authorized COVID-19 diagnostics, BioCentury, 2020, https://www.biocentury.com/article/304801/limits-of-detection-for-fda-authorized-covid-19-diagnostics</ref> Detection limits are often expressed in TCID50/mL. Copies/TCID50 for H1N1 is 2381 copies/TCID50 so 1 TCID50/mL represents 2381 copies per mL. It is a very rough method to determine concentration and it has a standard deviation of about 1048 copies/TCID50.<ref name="Arti33">Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses, PLoS One, 2015,Parker J, Fowler N, Walmsley ML, Schmidt T, Scharrer J, Kowaleski J, Grimes T, Hoyos S, Chen J., https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646456/</ref></sub></div>
  
 +
CLIA waived tests are tests cleared for POC setting. These are simple tests with a relatively low risk for an incorrect result. Tests are scored by seven criteria on a scale of one to three, where a score of 1 indicates the lowest level of complexity. The tests may be called CLIA waived, if the score is low enough. The seven criteria are:<ref name="Arti27">Categorization Criteria CLIA test, FDA, 2020, Inc., https://www.fda.gov/node/365445#scorecard</ref>
  
  
 +
1. Knowledge: scientific and technical knowledge required to use the test.
  
−
{| class="wikitable" style="margin-bottom:0"
+
2. Training and experience: training needed to perform the test.
−
!Company
+
 
−
!Product
+
3. Reagents and materials preparation: stability of materials and material preparation.
−
!Test name
+
 
−
!Sample Volume (μL)
+
4. Characteristics of operational steps: complexity of operational steps.
−
!Reportable range
 
−
!Dilution
 
−
!Precision
 
−
!Incubation time
 
−
!Measuring Technique
 
−
|-
 
−
|Beckman Coulter<ref name=”[23]”>Emit 2000 Valproic Acid Assay (2010, September) Retrieved from November 7, 2019''. Beckman.</ref> <ref name=”[24]”>Beckman Coulter system Reagent, AU400/AU400e (2012, February) Retrieved from November 7, 2019''. Beckman.</ref>
 
−
|AU2700/AU5400
 
−
|VALPROIC ACID EMIT® 2000
 
−
|3.5 μL
 
−
|< 150 μg/mL
 
−
|1:1
 
−
|Total CV < 4.3%<br /> Inter-assay: CV < 3.2%
 
−
|15-75 min.
 
−
|ELISA<sup>1</sup>
 
−
|-
 
−
|Roche COBAS<ref name=”[25]”>Cobas 8000 modular analyzer series (last update: 2019, November 8).</ref> <ref name=”[26]”>van Eckardstein, A et al. (2013). cobas 8000 Modular Analyzer Series Evaluated under Routine-like Conditions at 14 Sites in Australia, Europe, and the United States. Retrieved from October 23., at [https://pdfs.semanticscholar.org/beb9/eaea6d45ba8fec2f3049cc5e242d9d2cfb13.pdf
 
−
“https://pdfs.semanticscholar.org/beb9/eaea6d45ba8fec2f3049cc5e242d9d2cfb13.pdf
 
−
”].</ref>
 
−
|Cobas 8000
 
−
|Cobas 8000
 
−
|1.5–35
 
−
|3.15 – 150 mg/L <ref name=”[27]”>Therapeutic drug monitoring (2011). Retrieved from November 8, 2019.'' R-Biopharm AG.</ref>
 
−
|1:(3-121)
 
−
|Inter-assay: CV < 0.7% ~ 2.9%<br />Total CV<3%
 
−
|9 min – 27 min
 
−
|ELISA<sup>1</sup>
 
−
|-
 
−
|ABBOTT LABORATORIES DIAGNOSTIC DIVISION<ref name=”[28]”>iValproic Acid B1P350 (2009,August).</ref>
 
−
<ref name=”[29]”> Free Valproic Acid Assay (2014, April). Retrieved from November 3, 2019, at [https://www.cadth.ca/sites/default/files/pdf/lab-tests/06_Free_Valproic_Acid_Assay_e.pdf/“https://www.cadth.ca/sites/default/files/pdf/lab-tests/06_Free_Valproic_Acid_Assay_e.pdf”].</ref>
 
  
−
|B1P350
+
5. Calibration, quality control and proficiently testing materials: can be stable or labile.
−
|ARCHITECT iValproic Acid
 
−
|10
 
−
|2 - 150 μg/mL
 
−
|1:10
 
−
|Total CV≤ 7%
 
−
|29 min
 
−
|CMIA<sup>2</sup>
 
−
|-
 
−
|SIEMENS HEALTHCARE DIAGNOSTICS INC.<ref name=”[30]”> ADVIA 1900 Chemistry system</ref>
 
−
|67070
 
−
|ADVIA 1200 CHEMISTRYSYSTEM -VALPROIC ACID (VPA) ASSAY
 
−
|2-3
 
−
|NA
 
−
|1:5
 
−
|Inter-assay: CV = 0.2% - 5.3 %<br /> Total CV= 0.4%-5.3% <ref name=”[31]”> American Association for Clinical Chemistry, 70th AACC Annual Scientific Meeting (July, August, 2018). Retrieved from November 7, 2019</ref>
 
  
−
|3-21 min
+
6. Test system troubleshooting and equipment maintenance: the easiness and frequency of the troubleshooting and equipment maintenance.
−
|ELISA<sup>1</sup>
 
−
|-
 
−
|MICROGENICS CORPORATION<ref name=”[32]”>CEDIA Valproic Acid II Assay (2018, November). Retrieved from November 7, 2019</ref>
 
−
|62390
 
−
|CEDIA TDM ASSAY -VALPROIC ACID
 
−
|NA
 
−
|3.0-150 μg/mL
 
−
|NA
 
−
|Inter-assay: CV = 1.3% - 2.4 %<br /> Total CV= 1.8%-3.4%
 
−
|Reagent 1 : 2-5 min <br/>Reagent 2: 4-8 min
 
−
|ELISA<sup>1</sup>
 
  
 +
7. Interpretation and judgement: required to perform analytic processes and resolution of problems.
 +
 +
An alternative type of testing is with a ‘molecular assay’, which is more accurate than RITDs, because it detects viral RNA or nucleic acids in respiratory specimens. This type of testing includes rapid molecular assays, RT-PCR and other nucleic acid amplification tests. Molecular tests are mostly used in hospitals, some are able to detect both influenza A and B. Others can identify different subtypes. The ‘molecular assays’ are more accurate and sensitive than RIDTs, but the time-to-result may be several hours.<ref name="Arti28">Diagnosing Flu, CDC, 2020, https://www.cdc.gov/flu/symptoms/testing.htm</ref><ref name="Arti29">Information on Rapid Molecular Assays, RT-PCR, and other Molecular Assays for Diagnosis of Influenza Virus Infection, CDC, 2020, https://www.cdc.gov/flu/professionals/diagnosis/molecular-assays.htm</ref>
  
 +
==Safety==
 +
When working with the influenza virus, influenza infection in humans can occur following a laboratory accident. For safety reasons, unbound hemagglutinin particles will be used in SensUs 2021, as a substitute for infectious influenza virus particles.
  
−
|}<div style="margin-bottom:1em"><sub>''1 ELISA: Enzyme Linked Immunosorbent Assay''<br />Note: 2.CMIA: Chemiluminescent Microparticle Immunoassay
+
===Lab protocols===
−
</sub></div>
+
The use of safety equipment combined with good practices is fundamental to laboratory safety and in helping to reduce the risks involved in dealing with biosafety hazards. Therefore, it is important that you consult with your local biosafety officer and comply to the safety rules of your own organization.
  
−
To measure unbound VPA, blood samples are treated by ultrafiltration, followed by an immunoassay, also referred to as ELISA. By performing the ultrafiltration, the protein-bound form of VPA is separated from its unbound form. After that, the level of the unbound fraction can  be measured by an immunoassay, e.g. an ELISA kit. Alternatively, LC-MS can be used to measure VPA.<ref name=”[40]”> ao, S., Miao, H., Tao, X., Jiang, B., Xiao, Y., Cai, F., … Chen, W. (2011, July 1). LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma, Journal of Chromatography B
+
According to Article 4.84 of the Working Conditions Decree in The Netherlands, inactivated virus particles can be classified as category 1 of the biological agents<ref name="Arti30">1Short manual for the ML-I, ML-II laboratory(gmo-labs) and/or laboratory for working with biological agents / human materials, University of Twente, 2018,https://www.utwente.nl/.uc/f4fcab199010209618400ac90d20284e61d13c337a78900/short-manual-gmo.pdf</ref>, as they are unlikely to cause disease in humans. Therefore, the biosafety guidelines as defined in the ML-1 (minimum containment level) have to be followed. It is stipulated in the guidelines that general laboratory protocols such as the usage of barrier protection (lab coats, gloves, and face protection) when handling the samples are to be followed. The protection barriers are not always necessary from a microbiological perspective. However, it is compulsory when handling disinfectants or solvents.
−
Volume 879, Retrieved November 28, 2019, at [https://www.sciencedirect.com/science/article/pii/S1570023211003278?via=ihub“https://www.sciencedirect.com/science/article/pii/S1570023211003278?via=ihub”].</ref>
 
−
Steps such as solvent extraction or derivation must be executed prior to a HPLC assay, which takes significant time<ref name=”[29]”> Free Valproic Acid Assay (2014, April). Retrieved from November 3, 2019, at [https://www.cadth.ca/sites/default/files/pdf/lab-tests/06_Free_Valproic_Acid_Assay_e.pdf/“https://www.cadth.ca/sites/default/files/pdf/lab-tests/06_Free_Valproic_Acid_Assay_e.pdf”].</ref>
 
−
. CMIA is a special type of ELISA <ref name=”[34]”> Ilyas M., Ahmad. I (2014, July 12), Chemiluminescent microparticle immunoassay based detection and prevalence of HCV infection in district Peshawar Pakistan, third alinea Background, Retrieved from November 7, 2019, at [https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-11-127“https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-11-127”].</ref>
 
−
.
 
−
Several innovations are being investigated for VPA testing. For example, 2D-LC system (two-dimensional chromatography) was studied, allowing large volume injection, reducing interfering components, and reducing the analysis time and preventing most interference components by selecting useful sections in the “heart-cut” column(1D) from entering the analysis column (2D).<ref name=”[11]”>  Liu, W., Shang, X., Yao, S., & Wang, F. (2019, August 20). A novel and nonderivatization method for the determination of valproic acid in human serum by two‐dimensional liquid chromatography. Retrieved October 17, 2019, at [https://doi.org/10.1002/bmc.4695.“https://doi.org/10.1002/bmc.4695.”].</ref>
 
−
. Another example is dried blood spot (DBS) followed by gas chromatography mass spectrometry (GC–MS), which does not require solvent extraction or elution. The limit of quantitation was 200 ng/mL. <ref name=”[12]”> Guo, M., Shao, L., Chen, X., Li, H., Wang, L., Pan, Y., & Tang, D. (2019, September 13). Assay of dried blood spot from finger prick for sodium valproate via ink auxiliary headspace gas chromatography mass spectrometry, Journal of Chromatography A Vol. 1601 p. 335-339. Retrieved October 24, 2019, at [ https://doi.org/10.1016/j.chroma.2019.05.039“ https://doi.org/10.1016/j.chroma.2019.05.039”].</ref>
 
  
 
== References ==
 
== References ==
  
 
<references />
 
<references />

Latest revision as of 15:20, 29 January 2021

Structure of the influenza virus.[1]

General information

The theme of SensUs 2021 is acute respiratory viruses. The current Covid-19 pandemic has made it apparent that large virus outbreaks can cause immense harm to human health and can disrupt society as a whole. One of the most common respiratory viruses is influenza A. Therefore, the influenza virus serves as an interesting research topic for this year’s Competition.

The influenza A virus is known to cause influenza in birds and some mammals, like humans. Several subtypes of the influenza A virus have been isolated from wild birds. Occasionally, viruses can be transmitted from wild birds to domestic animals, like chickens or pigs. This may give rise to human influenza.[2]

Influenza A viruses are negative-sense, single-stranded RNA viruses. Subtypes of influenza A are characterized by proteins on the outermost membrane of the virus, called hemagglutinin (H or HA) and neuraminidase (N or NA). H and N are the antigens of the virus, and play an important role in the interaction between the host’s immunological response and the virus. Recently, researchers have reported the discovery of an antibody that is generally effective against all subtypes of the influenza A virus.[3] Hemagglutinin will serve as this year's biomarker. In infected patients, bound hemagglutinin indicates the presence of the influenza virus. In the Testing Event, unbound hemagglutinin proteins will be used to mimic the immunologic response of the virus.

The subtype which will be used in SensUs 2021 is H1N1. Historically, H1N1 has been responsible for most deaths due to influenza. It is a popular influenza strain for research purposes.[4][5][6] Due to its popularity among researchers, antigens and antibodies are commercially available, making H1N1 suitable as a target for the SensUs Competition. Influenza A vaccines for humans have been developed. New versions of the vaccines are developed twice per year for use all over the world, which is necessary due to rapid mutations of the influenza virus. Every year during flu season, a large part of the population is vaccinated in order to protect individuals against the virus. However, due to unforeseen mutations of the virus, it might be possible that in a certain year a vaccine will prove ineffective. In that case large portions of the population would be at risk and a pandemic could occur. The probability of a major influenza A pandemic is estimated to be around 0.5-1% each year.[7]

History of influenza (A)

A lack of data up until 1500 AC complicates the research on influenza before that period.[8] Possibly the first influenza pandemic occurred around 6000 BC in China. The symptoms of human influenza seem to have been clearly described by Hippocrates, roughly 2,400 years ago[9] Although the virus seems to have caused epidemics throughout human history, historical data on influenza is difficult to interpret, due to the fact that symptoms of influenza are similar to those found in other respiratory diseases, like respiratory syncytial virus (RSV).

The most infamous and lethal outbreak was the 1918 flu pandemic, which lasted until 1920. The number of deaths is unknown, but estimates range from 17 to 100 million people.[10]. This pandemic has been described as "the greatest medical holocaust in history"[11] and may have killed as many people as the plague (Black Death). This huge death toll was caused by an extremely high infection rate of up to 50% and the severity of the symptoms, suspected to be caused by cytokine storms in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules called cytokines. One of the most recent outbreaks of influenza was the 2009 Swine Flu. Similar to the Spanish Flu, it was also of the subtype H1N1. The death toll of the 2009 pandemic is estimated to be around 150,000 to 575,000.[12]

Structure of the influenza virus

The virus particle (virion) is 80–120 nanometers in diameter.[13] The virion shape can be spherical, elliptical, or even filamentous with a length of tens of micrometers[13]. The virion is made up of a viral envelope containing two main types of proteins, wrapped around a central core. The two large proteins found on the outside of viral particles are hemagglutinin (HA) and neuraminidase (NA). HA is a protein that mediates binding of the virion to target cells and entry of the viral genome into the target cell, and therefore plays an important role in infecting healthy cells. NA is involved in releasing the progeny viruses once a cell has been infected and has started producing the virus itself.[14]

These two proteins are a target of interest for antiviral drugs.[15] Furthermore, they are also the antigen proteins to which a host antibodies can bind and trigger an immune response. Influenza type A viruses are categorized into different subtypes, or strains, based on which type of these two proteins is present on the surface of the virion. Currently, there are 16 subtypes of HA and 9 subtypes of NA known to exist. The most prevalent form of the different subtypes is H1N1. Single hemagglutinin-neuraminidase proteins, in which both HA and NA are found in a single protein, also exist. However, these will not be used in SensUs 2021.

Mechanism of infection

An influenza virus infecting a cell.[16]

The involvement of the hemagglutinin and neuraminidase proteins in the infection of H1N1 is essential for the virus reproduction, and a more specific mechanism is discussed below. The hemagglutinin protein (HA) has the role of searching for the sialic acid receptors in respiratory-lining cell membranes. Upon binding of this protein and the receptor, fusion of the virus and the cell membrane is facilitated with the help of glycan proteins. The virus then enters the cell where it sheds its shell and approaches the cell’s nucleus. Using the host replication mechanisms, copies of the virus are made. At this step of the infection process, important viral proteins are synthesized. These newly replicated viral elements subsequently leave through the cell membrane and infect other cells. To inhibit the exit of the viral components, sialic acid receptors on the cell can bind the HA glycoproteins. This is where viral evolution/mutability can play a role in the expansion of the capabilities of the virus. The neuraminidase glycoprotein (N) has the role of cleaving the sialic acid receptors, allowing the exit of the viral components which then go in search of a new host. After infection is complete, the H1N1 virus triggers cell apoptosis, leading to the death of the cell and spread of the virions.

Medical application

The influenza virus spreads when people with the flu cough, sneeze or talk. Infected people transfer tiny droplets to people close to them and infect them by the handover of droplets. During an outbreak, it is important to be able to control and prevent the virus from spreading further, for example, by implementing control measures. Those control measures can be determined based on the reproduction number. The reproduction number is defined as: “the expected number of secondary cases produced by a single infection in a completely susceptible population”.[17] A clear overview of the number of infected people is needed to calculate the reproduction number.

For disease control, it is crucial to know the infection rate in a specific region. The results may influence critical decisions such as whether to perform other diagnostic testing or to implement infection prevention and control measures for influenza. Furthermore, manpower can be in short supply during a pandemic and the speed at which someone can be tested and receive the result is of vital importance.

Biosensors that will be developed in SensUs 2021 are envisioned to be used outside the hospital in a point-of-care (POC) setting. Inside the hospital there is not a big advantage, as there are already very specific and accurate tests available for that setting.[18][19] Important applications are fast testing at the general practioner and at home. Therefore the test should be easy to use. The biosensors will be designed to enable a fast yes/no answer, based on measuring the concentration of hemagglutinin particles in the sample. The biosensors will not distinguish between different virus subtypes, as the subtype causing the pandemic is assumed to be known.

State of the Art

There are various influenza tests available on the market. The most common tests are ‘rapid influenza diagnostic tests’, also called RIDTs. RIDTs provide results in a qualitative way within approximately 10-15 minutes and work by detecting the parts of the virus that stimulate an immune response.[20] The table contains examples of available rapid influenza diagnostic tests that provide results in 10-15 minutes. In the SensUs Competition, a saliva based test will be developed. Moreover, SensUs strives to stimulate developement of sensors that provide results within 5-10 minutes and that are as sensitive as possible. The SensUs Competition aims to innovate the field of influenza biosensing by using saliva as a matrix, by improving the speed of the test, and by targeting a high sensitivity.

Manufacturer Product CLIA Waived Platform Sensitivity

(PPA*)

Specificity

(NPA**)

Sample type LOD*** (H1N1)

TCID50/mL****

Abbott[21] Binax Now Influenza A & B Card 2 Yes DIGIVAL 70-89% 90-99% NPS, NS direct N/A
Becton Dickinson & Co.[22] BD Veritor™ Flu A + B Yes BD veritor Reader 82% 98% NPS, NS direct 3.3*102-5.0*103
Becton Dickinson & Co.[22] BD Veritor™ Flu A + B Yes BD veritor plus Analyzer 83,6% 97,5% NPS, NS direct 3.3*102-5.0*103
Quidel Corp.[23] Sofia® Influenza A + B FIA Yes Sofia(2) FIA Analyzer 90-99% 95-96% NS, NPS, NPA, NPW direct, NP, NPA, NPW in VTM N/A
Quidel Corp[24] QuickVue® Influenza A + B Yes N/A 81,5% 97,8% NS, NPS, NPA, NPW direct, NP, NPA, NPW in VTM 1.63*103-4.4*103
Princeton BioMeditech Corp.[25] BioSign® Flu A & B

LABSCO

Yes N/A 89,2% 99,4% NS, NPS direct, NPA(waived), NPW(not waived) N/A
Remel/Thermo Fisher.[26] XPECT™ Flu A & B

LABSCO

No N/A 89-100% 100% NW, NS 1.63*103-4.41*103
Sekisui Diagnostics, [27] Acucy Influenza A&B Test Yes The Acucy System 96,4% 96,0% NPS, NS direct 1.4*101
Sekisui Diagnostics, [28] OSOM Ultra Plus Flu A&B Test Yes N/A 90,2-92,2% 75,1-85,7% NPS, NPA, NPW, NS 1.05*102
Access Bio, Inc., [29] CareStart Flu A&B Plus No N/A 79,9% 98,4% NPS 5.0*103-9.6*103


Table 1: Different rapid diagnostic tests for infuenza A

*PPA: positive percent agreement (True positive / total true positive)

**NPA: negative percent agreement (True negative / total true negative)

***LOD: Limit of detection

****TCID50/mL represents the viral load at which 50% of cells are infected when a solution containing the virus is added to the cell culture.[30] Detection limits are often expressed in TCID50/mL. Copies/TCID50 for H1N1 is 2381 copies/TCID50 so 1 TCID50/mL represents 2381 copies per mL. It is a very rough method to determine concentration and it has a standard deviation of about 1048 copies/TCID50.[31]

CLIA waived tests are tests cleared for POC setting. These are simple tests with a relatively low risk for an incorrect result. Tests are scored by seven criteria on a scale of one to three, where a score of 1 indicates the lowest level of complexity. The tests may be called CLIA waived, if the score is low enough. The seven criteria are:[32]


1. Knowledge: scientific and technical knowledge required to use the test.

2. Training and experience: training needed to perform the test.

3. Reagents and materials preparation: stability of materials and material preparation.

4. Characteristics of operational steps: complexity of operational steps.

5. Calibration, quality control and proficiently testing materials: can be stable or labile.

6. Test system troubleshooting and equipment maintenance: the easiness and frequency of the troubleshooting and equipment maintenance.

7. Interpretation and judgement: required to perform analytic processes and resolution of problems.

An alternative type of testing is with a ‘molecular assay’, which is more accurate than RITDs, because it detects viral RNA or nucleic acids in respiratory specimens. This type of testing includes rapid molecular assays, RT-PCR and other nucleic acid amplification tests. Molecular tests are mostly used in hospitals, some are able to detect both influenza A and B. Others can identify different subtypes. The ‘molecular assays’ are more accurate and sensitive than RIDTs, but the time-to-result may be several hours.[18][19]

Safety

When working with the influenza virus, influenza infection in humans can occur following a laboratory accident. For safety reasons, unbound hemagglutinin particles will be used in SensUs 2021, as a substitute for infectious influenza virus particles.

Lab protocols

The use of safety equipment combined with good practices is fundamental to laboratory safety and in helping to reduce the risks involved in dealing with biosafety hazards. Therefore, it is important that you consult with your local biosafety officer and comply to the safety rules of your own organization.

According to Article 4.84 of the Working Conditions Decree in The Netherlands, inactivated virus particles can be classified as category 1 of the biological agents[33], as they are unlikely to cause disease in humans. Therefore, the biosafety guidelines as defined in the ML-1 (minimum containment level) have to be followed. It is stipulated in the guidelines that general laboratory protocols such as the usage of barrier protection (lab coats, gloves, and face protection) when handling the samples are to be followed. The protection barriers are not always necessary from a microbiological perspective. However, it is compulsory when handling disinfectants or solvents.

References

  1. ↑ Host Protective Immune Responses against Influenza A Virus Infection, MDPI, 2020, Hi Eun Jung, Heung Kyu Lee, https://www.mdpi.com/1999-4915/12/5/504/htm
  2. ↑ Transmission of Avian Influenza A Viruses Between Animals and People, CDC, 2015, https://www.cdc.gov/flu/avianflu/virus-transmission.htm
  3. ↑ Super antibody' fights off flu, BBC, 2011, James Gallagher, https://www.bbc.com/news/health-14324901
  4. ↑ Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection, Research Gate, 2020, Sun, Honglei & Xiao, Yihong & Liu, Jiyu & Wang, Dayan & Li, Fangtao & Wang, Chenxi & Li, Chong & Zhu, Junda & Song, Jingwei & Sun, Haoran & Zhimin, Jiang & Liu, Litao & Zhang, Xin & Wei, Kai & Dongjun, Hou & Pu, Juan & Sun, Yipeng & Tong, Qi & Bi, Yuhai & Liu, Jinhua https://www.researchgate.net/publication/342555087_Prevalent_Eurasian_avian-like_H1N1_swine_influenza_virus_with_2009_pandemic_viral_genes_facilitating_human_infection
  5. ↑ Comparison of Hospitalized Patients With ARDS Caused by COVID-19 and H1N1, CHEST, Xiao Tang, Rong-Hui Du, Rui Wang, Tan-Ze Cao, Lu-Lu Guan, Cheng-Qing Yang, Qi Zhu, Ming Hu, Xu-Yan Li, Ying Li, Li-Rong Liang, Zhao-Hui Tong, Bing Sun, Peng Peng, Huan-Zhong Shi, 2020, https://www.sciencedirect.com/science/article/pii/S0012369220305584
  6. ↑ Landscape of coordinated immune responses to H1N1 challenge in humans, Journal of Clinical Investigation, 2020, Zainab Rahil, Rebecca Leylek, Christian M. Schürch, Han Chen, Zach Bjornson-Hooper, Shannon R. Christensen, Pier Federico Gherardini, Salil S. Bhate, Matthew H. Spitzer, Gabriela K. Fragiadakis, Nilanjan Mukherjee, Nelson Kim, Sizun Jiang, Jennifer Yo, Brice Gaudilliere, Melton Affrime, Bonnie Bock, Scott E. Hensley, Juliana Idoyaga, Nima Aghaeepour, Kenneth Kim, Garry P. Nolan, David R. McIlwain https://www.sciencedirect.com/science/article/pii/S0012369220305584
  7. ↑ Pandemic risk: how large are the expected losses?, WHO, 2017, Victoria Y Fan, Dean T Jamisonb & Lawrence H Summers, https://www.who.int/bulletin/volumes/96/2/17-199588.pdf
  8. ↑ Internet‐Based Intelligence in Public Health Emergencies, NATO Science for Peace and Security Series - E: Human and Societal Dynamics, 2013 Mordini E., Green M., https://www.iospress.nl/book/internet%E2%80%90based-intelligence-in-public-health-emergencies/
  9. ↑ 2,500-year Evolution of the Term Epidemic, Emerging infectious diseases, 2006, Martin PM, Martin-Granel E, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373038/
  10. ↑ Reassessing the Global Mortality Burden of the 1918 Influenza Pandemic, American journal of epidemiology, 2018, Spreeuwenberg P, Kroneman M, Paget J, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314216/
  11. ↑ Reviewing the History of Pandemic Influenza: Understanding Patterns of Emergence and Transmission , Pathogens, 2016, Saunders-Hastings PR, Krewski D, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198166/
  12. ↑ 2009 H1N1 Pandemic (H1N1pdm09 virus), centers for disease control and prevention, 2010, https://www.cdc.gov/flu/pandemic-resources/2009-h1n1-pandemic.html
  13. ↑ 13.0 13.1 Native Morphology of Influenza Virions, Frontiers in microbiology, 2011, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249889/
  14. ↑ Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase, Virology journal , 2013, Cohen M, Zhang XQ, Senaati HP, Chen HW, Varki NM, Schooley RT, Gagneux P, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842836/
  15. ↑ Recent Strategies in the Search for New Anti-Influenza Therapies, Current Drug Targets , 2003, J.C. Wilson, M. von Itzstein, https://www.eurekaselect.com/63785/article
  16. ↑ Sugars on Cell Surface Are Key to Flu Infections, National Institute of Health, 2008, https://www.nih.gov/news-events/nih-research-matters/sugars-cell-surface-are-key-flu-infections
  17. ↑ Notes on R0, 2007, James Holland Jones, https://web.stanford.edu/~jhj1/teachingdocs/Jones-on-R0.pdfe
  18. ↑ 18.0 18.1 Diagnosing Flu, CDC, 2020, https://www.cdc.gov/flu/symptoms/testing.htm
  19. ↑ 19.0 19.1 Information on Rapid Molecular Assays, RT-PCR, and other Molecular Assays for Diagnosis of Influenza Virus Infection, CDC, 2020, https://www.cdc.gov/flu/professionals/diagnosis/molecular-assays.htm
  20. ↑ Rapid Influenza Diagnostic Tests, 2016, Centers for Disease Control and Prevention, https://www.cdc.gov/flu/professionals/diagnosis/clinician_guidance_ridt.htm
  21. ↑ Abbott RIDT Product Information, Abbott, https://www.globalpointofcare.abbott/nl/product-details/binaxnow-influenza-a-and-b.html
  22. ↑ 22.0 22.1 Becton Dickinson & Co. RIDT Product Information, Becton Dickinson & Co., https://www.bd.com/en-us/offerings/capabilities/microbiology-solutions/point-of-care-testing/veritor-system
  23. ↑ Quidel RIDT Product Information, Quidel, https://www.quidel.com/immunoassays/rapid-influenza-tests/sofia-influenza-fia
  24. ↑ Quidel QuickVue RIDT Product Information, Quidel, https://www.quidel.com/sites/default/files/product/documents/EF1350313EN00_1.pdf
  25. ↑ BioSing RIDT Product Information, Princeton BioMeditech Corp., http://punchout.medline.com/product/BioSign-Rapid-Flu-AB-Antigen-Panel-Test-by-Princeton-BioMeditech/Influenza-Testing/Z05-PF176440?question=&index=P11&indexCount=11#mrkSpec
  26. ↑ XPECT™ RIDT Product Information, Remel/Thermo Fisher, https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FMBD%2FInstructions%2FIFU24600%2520.pdf&title=WHBlY3QgRmx1IEEgYW5kIEI=
  27. ↑ Acucy Influenza A&B Test Product Information, Sekisui Diagnostics, https://www.sekisuidiagnostics.com/wp-content/uploads/2019/08/Acucy-Influenza-AB-Package-Insert.pdf
  28. ↑ OSOM Ultra Plus Flu A&B Test Product Information, Sekisui Diagnostics, https://www.sekisuidiagnostics.com/wp-content/uploads/2019/04/OSOM_P-52631-D__web.pdf
  29. ↑ CareStart Flu A&B Plus Product Information, Access Bio, Inc., https://www.accessdata.fda.gov/cdrh_docs/pdf19/K191514.pdf
  30. ↑ Limits of detection for FDA-authorized COVID-19 diagnostics, BioCentury, 2020, https://www.biocentury.com/article/304801/limits-of-detection-for-fda-authorized-covid-19-diagnostics
  31. ↑ Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses, PLoS One, 2015,Parker J, Fowler N, Walmsley ML, Schmidt T, Scharrer J, Kowaleski J, Grimes T, Hoyos S, Chen J., https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646456/
  32. ↑ Categorization Criteria CLIA test, FDA, 2020, Inc., https://www.fda.gov/node/365445#scorecard
  33. ↑ 1Short manual for the ML-I, ML-II laboratory(gmo-labs) and/or laboratory for working with biological agents / human materials, University of Twente, 2018,https://www.utwente.nl/.uc/f4fcab199010209618400ac90d20284e61d13c337a78900/short-manual-gmo.pdf
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