Case study for COVID-19 -

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Maria B. Lazebnik, Ph.D., Bentley University, Department of Natural and Applied Sciences Case Study To Test or Not to Test: The Ins and Outs of COVID-19 Testing Background : On the March 12, 2020, the WHO (World Health Organization) has designated coronovirus-induced pneumonia, known as "coronavirus disease 2019" (abbreviated COVID-19), as a pandemic, which generally means that it has spread to more than one continent (1). The virus has been named “SARS-CoV-2” and the disease it causes is COVID-19. The novel outbreak of coronovirus disease was first reported in Wuhan, China in December 2019, and has spread worldwide including the US (2). SARS-CoV-2 virus is related to SARS and MERS, which have caused epidemics in the past. Additionally, COVID- 19 seems to be a zoonotic disease, which is transmitted from an animal host to human. Based on recent genomic data, it is hypothesized that the virus originated in bats, then jumped to Pangolins, which are used in Chinese medicine and are valued for their meat, and ended up being transmitted to human hosts. COVID-19 is spread person-to-person via respiratory droplets produced when a person coughs or sneezes. Typical clinical symptoms include fever, dry cough, breathing difficulties (dyspnoea), headache and pneumonia (2). While there is no treatment specifically approved for COVID-19, there are many clinical trials being conducted to develop medicines for COVID-19. Additionally there a vaccine trial underway for COVID-19 (3). Clinical diagnosis is based on several factors including epidemiological history, clinical symptoms, laboratory tests described below, chest CT scans, and a blood culture. There are two approaches to detecting the presence of SARC-CoV2 virus in the lab. One is based on detection of the viral nucleic acid, and another is based on detecting the immune response to the virus. Methods based on Nucleic Acid Detection: Laboratory diagnosis is mainly based on using real-time reverse transcription polymerase chain reaction (RT-qPCR), where "q" stands for quantitative, meaning that the amount of the nucleic acid is being quantified and measured in real time. RT-qPCR detects the presence of SARS-CoV-2 virus' nucleic acid material in respiratory samples, such nasopharyngeal swab or sputum, which can be obtained from a patient's nasal cavity or throat, or saliva. Since the virus genetic material is RNA, reverse transcriptase (RT) is first used to copy the RNA into cDNA for the PCR test. Then a fluorescent dye, such as SYBR green, is used to bind to the PCR product, and allows to measure the amount of generated fluorescent PCR product in real time. High-Throughput sequencing may also be used an alternative method, but it is more expensive and the equipment is limited to larger hospitals and testing centers. Methods based on Immune Response Identification Technology : Point-of-care Testing (POCT) and enzyme-linked immunosorbent assay (ELISA) are tests, which allows for detection of IgM/IgG antibodies, which are usually produced by a patient

Maria B. Lazebnik, Ph.D., Bentley University, Department of Natural and Applied Sciences in response to a viral infection. However, antibodies may not be detectable until a week or longer after infection, so RT-qPCR is preferred early in an infection. --------------------------------------------------- Part 1: Dr. Matt Sims, a physician at a hospital in Michigan observed four cases of what appeared to be a viral pneumonia within the last week. The symptoms resemble those associated with COVID- 19. The patients all had high fevers and difficulty breathing, and their x-rays showed signs of pneumonia. Bacterial cultures were negative. None of the patients reported traveling out of the country recently. Two of the patients have a relative who recently attended a Biogen Conference in Massachusetts, which has been linked to approximately 100 cases of COVID-19. The first patient who was admitted seems to be recovering, but the second patient has taken a turn for the worse, and may not last the night. Dr. Sims and the rest of the clinical staff have been taking every possible precaution, and the patients are in an isolation ward in the hospital. Dr. Simms is anxiously awaiting the results of the laboratory tests. To analyze this case, two types of tests are necessary. Dr. Sims runs an ELISA test on blood samples to test for the presence of COVID-19 IgG antibody. Also, because COVID-19 is an RNA virus, Dr. Sims decides to run RT-qPCR on the samples to try to detect the virus genetic material; Then the c-DNA from these samples is amplified and used in the real-time PCR test with SYBR green fluorescent dye. Samples include: Blood Serum and Nucleic Acid samples: Positive control for CoV-19 Negative control Patient 1 (37-year-old female) Patient 2 (67-year old male, Dad of patient 1) Patient 3 (43-year-old female) Patient 4 (88-year old male) Fig 1: shows Real-time PCR results. In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (ie exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (ie the lower the Ct level the greater the amount of target nucleic acid in the sample). PCR assays undergo 40 cycles of amplification.  Ct < 29 are strong positive reactions indicative of abundant target nucleic acid in the sample  Ct of 30-37 are positive reactions indicative of moderate amounts of target nucleic acid

Maria B. Lazebnik, Ph.D., Bentley University, Department of Natural and Applied Sciences  Ct of 38-40 are weak reactions indicative of minimal amounts of target nucleic acid , background signal Samples Ct value A Negative control sample 37 B Positive control sample 19.88 C Patient 1 23.75 D Patient 2 17.9 E Patient 3 21 F Patient 4 38.5 Fig2: shows ELISA results. Blue color in the well represents that COVID-19 viral protein is detected in the sample. Questions: 1. Based on the PCR and ELISA results, are any of the patients infected with the COVID-19 coronavirus? List the results for each patient. (3 points) a. Patient 1 (Sample C): - PCR: Ct value of 23.75, so it is positive - ELISA test also came back positive because it is indicated with the blue color. Conclusion: The patient tested POSITIVE for Covid-19 b. Patient 2 (Sample D):

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Maria B. Lazebnik, Ph.D., Bentley University, Department of Natural and Applied Sciences - PCR: Ct value of 17.9, so it is positive - ELISA test also came back positive because it is indicated with the blue color. Conclusion: The patient tested POSITIVE for Covid-19 c. Patient 3 (Sample E): - PCR: Ct value of 17.9, so it is positive - ELISA test could be a false negative result for the viral protein of COVID-19 Conclusion: Since the patient has a strong positive PCR result, the patient most likely is infected of Covid-19. d. Patient 4 (Sample F): - PCR: Ct value of 38.5, since it is a weak response, it can be a false positive or negative. - ELISA test came back as negative since it is not indicated with the blue color. Conclusion: Probably this patient is NEGATIVE for Covid-19 or could have low viral. 2. Why did the doctor use the RT-PCR test instead of just using PCR? (hint: think about the function of the RT enzyme) (2 points) - The doctor used the RT-PCR because the Covid19 is because of an RNA virus. The PCR studies the DNA so the RT converts the RNA from the virus before it can be amplified by the PCR. This process needs to be done since the PCR cannot amplify right away the RNA. 3. How might these patients have been exposed to the virus? (1 point) - These patients could have been exposed to the virus through whom attended the Biogen Conference that is related to the COVID-19 cases through its community transmission since that they have not travel out from the country. 4. If any of the patients are negative for COVID-19, what else could be causing their symptoms? (2 points) - If any patient like patient 4, that came out negative for Covid-19 can share similar symptoms, but it could have contracted other infections like influenza, or other viral pneumonias. Other tests can be done to determine what type of infection the patient has.

Maria B. Lazebnik, Ph.D., Bentley University, Department of Natural and Applied Sciences References: Adapted and modified SARS case A. Engaging Students in Molecular Biology via Case-Based Learning By M Bergland, K Klyzcek et. al. Science 27 JUL 2012 : 426-427 https://www.cdc.gov/coronavirus/2019-ncov/about/testing.html https://www.cdc.gov/coronavirus/2019-nCoV/lab/index.html

Case study for COVID-19 -

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