RT-qPCR investigation of post-mortem tissues during COVID-19.

Publisher: Elsevier Sp z.o.o Country of Publication: Poland NLM ID: 101221755 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1214-0287 (Electronic) Linking ISSN: 1214021X NLM ISO Abbreviation: J Appl Biomed Subsets: MEDLINE

Publication: <2013-> : Wroclaw : Elsevier Sp z.o.o.
Original Publication: České Budějovice, Czech Republic : University of South Bohemia, Faculty of Health and Social Studies

COVID-19*/virology ; COVID-19*/diagnosis ; COVID-19*/genetics ; SARS-CoV-2*/genetics ; RNA, Viral*/genetics ; RNA, Viral*/analysis ; Autopsy*/methods; Humans ; Male ; Real-Time Polymerase Chain Reaction/methods ; Female ; Lung/virology ; Lung/pathology ; Lung/diagnostic imaging ; Middle Aged ; Aged ; COVID-19 Nucleic Acid Testing/methods ; Trachea/virology ; Trachea/pathology ; Trachea/diagnostic imaging ; Adult ; Nasopharynx/virology

In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results.
Competing Interests: The authors report no conflicts of interest in this work.

Abid MB, Chhabra S, Buchan B, Graham MB, Abedin S, Thapa B, et al. (2021). Bronchoalveolar lavage-based COVID-19 testing in patients with cancer. Hematol Oncol Stem Cell Ther 14(1): 65-70. DOI: 10.1016/j.hemonc.2020.09.002. (PMID: 3305878710.1016/j.hemonc.2020.09.002)
Agency for Strategic planning and reforms of the Republic of Kazakhstan, Bureau of National statistics (2020). [online] [cit. 2020-08-02]. Available from: https://old.stat.gov.kz/.
Al-Omari A, Rabaan AA, Salih S, Al-Tawfiq JA, Memish ZA (2019). MERS coronavirus outbreak: Implications for emerging viral infections. Diagn Microbiol Infect Dis 93(3): 265-285. DOI: 10.1016/j.diagmicrobio.2018.10.011. (PMID: 3041335510.1016/j.diagmicrobio.2018.10.011)
Amirouche A, Ait-Ali D, Nouri H, Boudrahme-Hannou L, Tliba S, Ghidouche A, Bitam I (2021). TRIzol-based RNA extraction for detection protocol for SARS-CoV-2 of coronavirus disease 2019. New Microbes and New Infections 41: 100874. DOI: 10.1016/j.nmni.2021.100874. (PMID: 3381580710.1016/j.nmni.2021.100874)
Beltempo P, Curti SM, Maserati R, Gherardi M, Castelli M (2021). Persistence of SARS-CoV-2 RNA in post-mortem swab 35 days after death: A case report. Forensic Sci Int 319: 110653. DOI: 10.1016/j.forsciint.2020.110653. (PMID: 3336024210.1016/j.forsciint.2020.110653)
Bruce EA, Huang ML, Perchetti GA, Tighe S, Laaguiby P, Hoffman JJ, et al. (2020). Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step. PLOS Biology 18(10): e3000896. DOI: 10.1371/journal.pbio.3000896. (PMID: 3300698310.1371/journal.pbio.3000896)
Caniego-Casas T, Martínez-García L, Alonso-Riaño M, Pizarro D, Carretero-Barrio I, Martínez-de-Castro N, et al. (2022). RNA SARS-CoV-2 Persistence in the Lung of Severe COVID-19 Patients: A Case Series of Autopsies. Front Microbiol 13: 824967. DOI: 10.3389/fmicb.2022.824967. (PMID: 3517370110.3389/fmicb.2022.824967)
Chu DK, Pan Y, Cheng SM, Hui KP, Krishnan P, Liu Y, et al. (2020). Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia. Clin Chem 66(4): 549-555. DOI: 10.1093/clinchem/hvaa029. (PMID: 3203158310.1093/clinchem/hvaa029)
Clinical protocol for the diagnosis and treatment of coronavirus infection COVID-19 in adults. Ministry of Health of the Republic of Kazakhstan (2022). Protocol No. 166. [online] [cit. 2022-07-25]. Available from: https://online.zakon.kz/document/?doc_id=36043894&pos=6;-109#pos=6;-109.
Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al. (2020). Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25(3): 2000045. DOI: 10.2807/1560-7917.ES.2020.25.3.2000045. (PMID: 3199238710.2807/1560-7917.ES.2020.25.3.2000045)
COVID-19 Autopsy Project (2020). The first COVID-19 autopsy in Spain performed during the early stages of the pandemic. Rev Esp Patol 53(3): 182-187. DOI: 10.1016/j.patol.2020.05.004. (PMID: 3265096910.1016/j.patol.2020.05.004)
D'Cruz RJ, Currier AW, Sampson VB (2020). Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). Front Cell Dev Biol 8: 468. DOI: 10.3389/fcell.2020.00468. (PMID: 3258271810.3389/fcell.2020.00468)
D'Errico S, Zanon M, Montanaro M, Radaelli D, Sessa F, Di Mizio G, et al. (2020). More than Pneumonia: Distinctive Features of SARS-Cov-2 Infection. From Autopsy Findings to Clinical Implications: A Systematic Review. Microorganisms 8(11): 1642. DOI: 10.3390/microorganisms8111642. (PMID: 3311406110.3390/microorganisms8111642)
Ezhilan M, Suresh I, Nesakumarb N (2021). SARS-CoV, MERS-CoV and SARS-CoV-2: A Diagnostic Challenge. Measurement (Lond). 168: 108335. DOI: 10.1016/j.measurement.2020.108335. (PMID: 10.1016/j.measurement.2020.108335)
Filchakova O, Dossym D, Ilyas A, Kuanysheva T, Abdizhamil A, Bukasov R (2022). Review of COVID-19 testing and diagnostic methods. Talanta 244: 123409. DOI: 10.1016/j.talanta.2022.123409. (PMID: 3539068010.1016/j.talanta.2022.123409)
Freppel W, Merindol N, Rallu F, Bergevin M (2020). Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene Allplex™ 2019-nCoV assay. Virol J 17(1): 196. DOI: 10.1186/s12985-020-01468-x. (PMID: 3333953910.1186/s12985-020-01468-x)
Gaipov A, Gusmanov A, Abbay A, Sakko Y, Issanov A, Kadyrzhanuly K, et al. (2021). SARS-CoV-2 PCR-positive and PCR-negative cases of pneumonia admitted to the hospital during the peak of COVID-19 pandemic: analysis of in-hospital and post-hospital mortality. BMC Infect Dis 21(1): 458. DOI: 10.1186/s12879-021-06154-z. (PMID: 3401604310.1186/s12879-021-06154-z)
Gokulan CG, Kiran U, Kuncha SK, Mishra RK (2021). Temporal stability and detection sensitivity of the dry swab-based diagnosis of SARS-CoV-2. J Biosc 46: 95. DOI: 10.1007/s12038-021- 00216-9. (PMID: 10.1007/s12038-021-)
Hong KH, Lee SW, Kim TS, Huh HJ, Lee J, Kim SY, et al. (2020). Guidelines for Laboratory Diagnosis of Coronavirus Disease 2019 (COVID-19) in Korea. Ann Lab Med 40(5): 351-360. DOI: 10.3343/alm.2020.40.5.351. (PMID: 3223728810.3343/alm.2020.40.5.351)
Johns Hopkins Coronavirus Resource Center (2023). Mortality analysis. [online] [cit. 2023-06-07]. Available from: https://coronavirus.jhu.edu/data/mortality.
Kazakov SA, Balatskaya SV, Johnston BH (2006). Ligation of the hairpin ribozyme in cis induced by freezing and dehydration. RNA 12(3): 446-456. DOI: 10.1261/rna.2123506. (PMID: 1649523710.1261/rna.2123506)
KZ Health Ministry official page (2023). CoronaVirus2020KZ. [online] [cit. 2023-02-27]. Available from: https://www.coronavirus2020.kz.
Ma S, Huang Y, van Huystee RB (2004). Improved plant RNA stability in storage. Anal Biochem 326(1): 122-124. DOI: 10.1016/j.ab.2003.10.026. (PMID: 1476934510.1016/j.ab.2003.10.026)
Mallmann L, Hermann BS, Schallenberger K, Demoliner M, Eisen AKA, Heldt FH, et al. (2021). Proteinase K treatment in absence of RNA isolation classical procedures is a quick and cheaper alternative for SARS-CoV-2 molecular detection. J Virol Methods 293: 114131. DOI: 10.1016/j.jviromet.2021.114131. (PMID: 3379860610.1016/j.jviromet.2021.114131)
Muller PY, Janovjak H, Miserez AR, Dobbie Z (2002). Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 32(6): 1372-1374, 1376, 1378-1379.
Musso N, Falzone L, Stracquadanio S, Bongiorno D, Salerno M, Esposito M, et al. (2021). Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples. Diagnostics (Basel) 11(7): 1158. DOI: 10.3390/diagnostics11071158. (PMID: 10.3390/diagnostics11071158)
Nguyen PY, Chen XJ, Kunasekaran M (2020). Rise in pneumonia cases of unknown aetiology in Kazakhstan in June 2020: A rapid analysis. Global Biosecurity 1(4). DOI: 10.31646/gbio.81. (PMID: 10.31646/gbio.81)
Poloni TE, Moretti M, Medici V, Turturici E, Belli G, Cavriani E, Ceroni M (2022). COVID-19 pathology in the lung, kidney, heart and brain: The different roles of T-cells, macrophages, and microthrombosis. Cells 11(19): 3124. DOI: 10.3390/cells11193124. (PMID: 3623108710.3390/cells11193124)
Qamar W, Khan MR, Arafah A (2017). Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method. Saudi J Biol Sci 24(7): 1465-1469. DOI: 10.1016/j.sjbs.2016.09.016. (PMID: 3029421410.1016/j.sjbs.2016.09.016)
Sablone S, Solarino B, Ferorelli D, Benevento M, Chironna M, Loconsole D, et al. (2021). Post-mortem persistence of SARS-CoV-2: a preliminary study. Forensic Sci Med Pathol 17(3): 403-410. DOI: 10.1007/s12024-021-00375-z. (PMID: 3396396710.1007/s12024-021-00375-z)
Schaefer IM, Padera RF, Solomon IH, Kanjilal S, Hammer MM, Hornick JL, Sholl LM (2020). In situ detection of SARS-CoV-2 inlungs and airways of patients with COVID-19. Mod Pathol 33(11): 2104-2114. DOI: 10.1038/s41379-020-0595-z. (PMID: 3256184910.1038/s41379-020-0595-z)
Teymouri M, Mollazadeh S, Mortazavi H, Ghale-Noie ZN, Keyvani V, Aghababaei F, et al. (2021). Recent advances and challenges of RT-PCR tests for the diagnosis of COVID-19. Pathol Res Pract 221: 153443. DOI: 10.1016/j.prp.2021.153443. (PMID: 3393060710.1016/j.prp.2021.153443)
WHO (2018). Laboratory testing for Middle East respiratory syndrome coronavirus interim guidance (revised). [online] [cit. 2023-06-07]. Available from: http://apps.who.int/iris/bitstream/handle/10665/259952/WHO-MERS-LAB-15.1-Rev1-2018-eng.pdf?sequence=1.
WHO COVID-19 dashboard (2023). Number of COVID-19 cases reported to WHO. [online] [cit. 2023-06-07]. Available from: https://covid19.who.int.
Wiersinga WJ, Rhodes A, Cheng AC, Peacock SJ, Prescott HC (2020). Pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (COVID-19): a review. JAMA 324(8): 782-793. DOI: 10.1001/jama.2020.12839. (PMID: 3264889910.1001/jama.2020.12839)
Yegorov S, Goremykina M, Ivanova R, Good SV, Babenko D, Shevtsov A, et al. (2021). Epidemiology, clinical characteristics, and virologic features of COVID-19 patients in Kazakhstan: A nation-wide retrospective cohort study. Lancet Reg Health Eur 4: 100096. DOI: 10.1016/j.lanepe.2021.100096. (PMID: 3388045810.1016/j.lanepe.2021.100096)
Zhu Y, Wang L, Yin Y, Yang E (2017). Systematic analysis of gene expression patterns associated with postmortem interval in human tissues. Sci Rep 7(1): 5435. DOI: 10.1038/s41598-017-05882-0. (PMID: 2871043910.1038/s41598-017-05882-0)
Zhussupov B, Saliev T, Sarybayeva G, Altynbekov K, Tanabayeva S, Altynbekov S, et al. (2021). Analysis of COVID-19 pandemics in Kazakhstan. J Res Health Sci 21(2): e00512. DOI: 10.34172/jrhs.2021.52. (PMID: 3446563610.34172/jrhs.2021.52)

AP09259103 Kazakhstan Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan; COVID-19 Kazakhstan Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan

Keywords: COVID-19; Post-mortem tissues; RNA extraction; RT-qPCR; SARS-CoV-2

0 (RNA, Viral)

Date Created: 20240624 Date Completed: 20240624 Latest Revision: 20240624

20240624

10.32725/jab.2024.013

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