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.
==Mechanism of ActionInfection==
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. (figure _). 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, the virus makes copies of itself. At this step of the infection process, important viral proteins are synthesized. These newly replicated viral elements subsequently attempt to leave through the cell membrane and infect other cells. To inhibit the exit of the viral components, sialic acid receptors on the cell membrane attempt to 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.
'''pictures'''
===Medical Application===
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 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 (Influenza (Flu) 2019). 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”(https://web.stanford.edu/~jhj1/teachingdocs/Jones-on-R0.pdf). 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 (Overview of Influenza testing methods 2020). 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 (Diagnosis, 2020). Important applications are fast testing at the GP 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 viral particles in the sample. The biosensors will not distinguish between different virus subtypes, as the subtype causing the pandemic is assumed to be known.
===Efficacy and influence on hepatic functionState of the Art===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>. '''Still paste some tables'''
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>
==Number of patients==
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.
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>
==Medical use and TDM==
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.
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>
. The test should measure the free concentration (i.e. the concentration of unbound valproate) as this fraction is pharmacologically active.
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>
. 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.
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>
==Safety & Lab protocols==
===Safety===
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>
. 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===
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>
==State of the art==
{| class="wikitable" style="margin-bottom:0"
!Company
!Product
!Test name
!Sample Volume (μL)
!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
|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
|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>
|}<div style="margin-bottom:1em"><sub>''1 ELISA: Enzyme Linked Immunosorbent Assay''<br />Note: 2When working with the influenza virus, influenza infection in humans can occur following a laboratory accident. For safety reasons, inactivated virus particles will be used in SensUs 2021, as a substitute for infectious influenza virus particles.CMIA: Chemiluminescent Microparticle Immunoassay</sub></div>
To measure unbound VPA, blood samples are treated by ultrafiltration, followed by an immunoassay, also referred ==Lab Protocols==The use of safety equipment combined with good practices is fundamental to laboratory safety and in helping to as ELISAreduce the risks involved in dealing with biosafety hazards. By performing the ultrafiltration, the protein-bound form of VPA is separated from its unbound formAccording to Article 4. After that, the level 84 of the unbound fraction can be measured by an immunoassayWorking Conditions Decree, e.g. an ELISA kit. Alternatively, LC-MS the inactivated virus particles 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 classified as category 1). LC–MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasmathe biological agents, Journal of Chromatography BVolume 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 they are unlikely 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 cause disease 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://virologyjhumans.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 injectionTherefore, reducing interfering components, and reducing only the analysis time and preventing most interference components by selecting useful sections biosafety guidelines as defined in the “heartML-cut” column1 (1Dminimum containment level) from entering have to be followed. It is stipulated in the guidelines that general laboratory protocols such as the analysis column usage of barrier protection (2D).<ref name=”[11]”> Liulab coats, W., Shang, X., Yao, S., & Wang, F. (2019, August 20gloves and face protection). A novel and nonderivatization method for when handling 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) samples are to be 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 protection barriers are not always necessary from finger prick for sodium valproate via ink auxiliary headspace gas chromatography mass spectrometry, Journal of Chromatography A Vol. 1601 pa microbiological perspective. 335-339. Retrieved October 24However, 2019, at [ https://doi.org/10.1016/j.chroma.2019.05.039“ https://doi.org/10.1016/j.chroma.2019.05.039”]it is compulsory when handling disinfectants or solvents.</ref>
== References ==
<references />
