key: cord-0955366-7q0yiss7 authors: Mikuła-Pietrasik, Justyna; Książek, Krzysztof title: Aging correlates with lower threshold cycle values for RdRP/RdRP+S genes during molecular detection of SARS-CoV-2 date: 2021-07-15 journal: Exp Gerontol DOI: 10.1016/j.exger.2021.111361 sha: d8e3eb4d6e15987b516ca05771fc56ae3612797c doc_id: 955366 cord_uid: 7q0yiss7 RT-qPCR is the most reliable molecular method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we analyzed results of RT-qPCR obtained for 3044 patients diagnosed as SARS-CoV-2-positive using four different molecular tests utilizing five RNA sequences. The analysis showed that patients' age inversely correlates with threshold cycle (Ct) values for RdRP gene (LightMix® Modular Wuhan CoV RdRP-gene by Roche Diagnostics) and RdRP+S genes (MutaPLEX® Coronavirus RT-PCR kit by Immundiagnostic). At the same time, there was no correlation between age and Ct values for E, N, and ORF1ab genes. When patients were grouped by age, mean Ct values for RdRP gene in older patients were significantly lower compared with younger individuals. Collectively, our report indicates that older SARS-CoV-2-infected individuals exhibit higher viremia at diagnosis than younger patients, which may reflect impaired functioning of their immune response and predispose to more severe disease and worse prognosis. Laboratory medicine aimed at acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis allows identifying three separate indicators of the infection: viral RNA, antigens, and specific antibodies produced by immune cells. Due to the highest sensitivity of nucleic acid amplification tests (NAATs), the real-time (RT)-quantitative (q)PCR directed on the identification of SARS-CoV-2 RNA is commonly treated as the gold-standard in COVID19 diagnostics (Yuce et al., 2020) . During the RT-qPCR, the presence of SARS-CoV-2 nucleic acid is shown as the exponential (S-shaped) curve of amplification, arising from the point where the amplification curve crosses a threshold line. This intersection point, called a threshold cycle (Ct), is a unique feature of a given sample and thus it can be treated as a relative measure of a target quantity. Considering that the number of a template copies doubles with each cycle of amplification, lower Ct values indicate higher number of target copies and vice versa. In case of virologic diagnostics, when RT-qPCR is conducted under reproducible conditions, i.e. the same reagents, reaction volumes, and instruments, Ct values may indirectly reflect the magnitude of viral load (Tom and Mina, 2020) . From the beginning of the pandemic, older people were at a relatively high risk of severe COVID19 and bad prognosis. In fact, aged patients are the group with the highest mortality among all infected patients (Kang and Jung, 2020) . Most frequently, it is related to the presence of various age-related diseases, but it cannot be ruled out that an impaired functionality of their immune system, a natural feature of organismal aging (Montecino-Rodriguez et al., 2013) , also plays a role. In this study, we analyzed retrospectively results of RT-qPCR reactions for more than three thousand SARS-CoV-2-positive patients to verify if there exists any relationship between their age and Ct values at which the viral RNA started to generate a diagnostically relevant fluorescence. Nasopharyngeal swabs were taken from 3044 symptomatic and asymptomatic patients living in the Greater Poland Voivodeship, between May and October 2020 (Table 1 ). The samples were analyzed towards SARS-CoV-2 infection in the Poznan University of Medical Sciences Coronavirus Laboratory using RT-qPCR. Informations regarding patients do not include their clinical status as this parameter was not evaluated at the stage of diagnosis and the laboratory in which the RT-qPCR was conducted has no access to those data. Viral RNA was extracted using MGISP-960 (MGI Tech Co., Ltd., Shenzhen, China), Chemagic 360 (PerkinElmer, Waltham, MA, USA), and Maxwell ® RSC (Promega, Madison, WI, USA). The material was subjected to RT-qPCR using LightCycler® 480 (Roche Diagnostics, Indianapolis, IN, USA). The distribution of samples between three RNA extractors was random and depended on laboratory needs on a given day, whereas their distribution among tests was related to their availability on the market. Four different molecular tests approved and validated for SARS-CoV-2 diagnostics were used strictly in line with manufacturers' instructions (Table 2) . RT-qPCR reactions were conducted up to 45th cycle ( Fig. 1 ). All consumables (e.g. tips, plates, strips) were consistent with requirements of each assay and mostly provided by tests' manufacturers. Statistical analysis was performed using GraphPad Prism™ v.9.0 (GraphPad Software, San Diego, USA). The correlations were analyzed using the Spearman test. The means were compared with the Kruskal-Wallis test and Dunns test (comparison of all pairs of groups) as the post hoc. Statistical significance was acknowledged when the P value was less than 0.05. The means were additionally corrected for multiple comparisons by controlling the False Discovery Rate on the basis of two-stage step-up method of Benjamin, Krieger and Yekutieli test with a desired falsediscovery rate Q less than 0.05 as the criterion. Molecular diagnostic of SARS-CoV-2 using RT-qPCR is based on the detection of at least two targetsviral RNA sequencesof which, ideally, one region is specific and one is conserved to avoid crossreactivity with other endemic coronaviruses as well as potential genetic drift (Tang et al., 2020b) . Indeed, coronaviruses contain several genes that have been identified and employed as targets in SARS-CoV-2 diagnostics. They belong to two categories, that is replication-driving genes and those coding for structural proteins. The first group includes RNA-dependent RNA polymerase (RdRP), hemagglutinin-esterase (HE), and open reading frame 1a (ORF1a) and ORF1b, whereas envelope glycoproteins spike (S), envelope (E), transmembrane (M), helicase (Hel), and nucleocapsid (N) belong to the second group (Mathuria et al., 2020) . In this study we analyzed results of RT-qPCR obtained for SARS-CoV-2-positive patients using assays from four manufacturers, in which four pairs of genes were utilized: RdRP/E, ORF1ab/N, RdRP+S/E (a trigenic test), and ORF1ab (RdRP region)/N. Correlative analysis revealed that Ct values obtained for RdRP gene (LightMix® Modular Wuhan CoV RdRPgene by Roche Diagnostics) and RdRP+S pair of genes (MutaPLEX® Coronavirus RT-PCR kit by Immundiagnostic) remain in an inverse relationship with patients' age. The older the patients were, the lower were the Ct values. In case of remaining genes and assays, any correlation between Ct values and age was absent (Table 3 ). In order to find out, which age values particularly determine this relationship, patients were assigned according to their ages into ten decade-wide groups. The comparison of means showed that Ct values for RdRP gene in patients 41-50, 51-60, and 91-100 years old were significantly lower compared with patients 21-30 and 0-10 years old. In case of RdRP+S pair statistically significant differences were found between Ct values established for 91-100 years old and 0-10 and 21-30 years old patients. Noteworthy, the statistical power of the differences obtained was also confirmed by additional analysis of the False Discovery Rate, in which the determined Q values confirmed the truth of the discoveries, eliminating the risk of a false negative result (Table 4 ). On the other hand, discrepancies in the size of some age groups were responsible for the lack of statistically significant differences between them although the average Ct values would seem to indicate otherwise. However, this situation was unavoidable because of the random distribution of patients at different ages. Taking into account that Ct values may indirectly reflect the viral load in a given patient, our results suggest that older SARS-CoV-2infected individuals have higher viremia at diagnosis (Tom and Mina, 2020) , which may at least partly explain more severe course of the disease and worse prognosis (Karahasan Yagci et al., 2020) . A relationship between lower Ct values and higher clinical severity has already been found years ago in patients with various upper respiratory tract infections, caused by adenovirus, human metapneumovirus, and parainfluenza virus (Fuller et al., 2013) . Significantly lower Ct values for aged patients (80-89 years old) were previously described by Buchan et al., albeit six important differences with respect to our study should be emphasized. The first is that they analyzed three times smaller cohort of patients, other genes (ORF1 and E), employed only one molecular test, did not include a correlative analysis, andlast but not leastanalyzed the differences between means in a quite unique way, that is by comparing of average Ct values for each age group with the mean of all Ct values (Buchan et al., 2020) . At the same time, our findings challenge those by Heald-Sargent et al., who found that young children (<5 years old) have higher viral load than adults (18-65), but made this observation using far lower group of patients that we did (145 vs. 3044) and did not perform correlative tests (Heald-Sargent et al., 2020) . As per potential reasons of higher viral load in aged individuals and specific course of the disease in this group, one may speculate that they may be linked with age-associated changes in a functioning of immune system. These changes include declined adaptive immunity (immunosenescence) coupled with activated innate immunity (inflamm-aging) (Salminen et al., 2008) . Major features of immunosenescence include regression of thymus, decreased numbers of pro-and pre-B cells, reduced ability of hematopoietic stem cells to repopulate, diminished RAG expression, impaired cytotoxicity of NK cells, reduced phagocytic activity of neutrophils, reduced responsiveness of naïve T cells to interleukin 2 (IL-2), and decreased naïve to memory T cell ratio. All these changes contribute to a progressive deterioration of the ability of aged people to respond to infections (Aw et al., 2007) . At the same time, one may not exclude that the worsening of immune response in elderly individuals may also depend on some specific features of SARS-CoV-2 itself, including its plausible ability to modulate some regulatory mechanisms in the host (e.g. miRNAs) that facilitate the virus expansion (Bartoszewski et al., 2020) . On the other hand, at the level of tissues and organs, mostly due to the accumulation of senescent cells, several proinflammatory cytokines, chemokines, growth factors, and extracellular matrix remodeling molecules are overproduced generating a microenvironment that promotes various pathological changes, leading to increased morbidity and mortality of aged individuals (Xia et al., 2016) . Taking into account this dichotomic pattern of age-associated changes in the immunologic status one may suppose that the higher viremic load in this group of patients may be closely linked with immunosenescence phenomenon, whereas the inflamm-aging phenotype may synergize with cytokine storm recognized as the prime determinant of high mortality of COVID19 patients. Interleukin-6 (IL-6) is the best example of this plausible relationship, as its elevated level is one of the most reliable markers of inflamm-aging (Maggio et al., 2006) , andat the same timeits concentration in severely ill COVID19 patients is significantly higher than in those who experience moderate symptoms of the disease (Hojyo et al., 2020; Tang et al., 2020a) . In this regard we can, however, only speculate that cytokines overproduced by senescent cells constitute an undetermined part of the total amount of proinflammatory cytokines in aged patients. Taken together, results provided in this report show that some molecular tests, utilizing RT-qPCR detection of RdRP/RdRP+S gene, inform about relatively high SARS-CoV-2 viremia in aged individuals at diagnosis, which may directly translate to severity of the disease and its outcome on the basis of several age-associated changes in immunological status of these patients. Taking into account a well-established knowledge about mechanisms of inflamm-aging development, it seems reasonable to ask whether the use of senolytics that eliminate senescent (read: cytokine overproducing) cells from the organism (Hickson et al., 2019) could be a barrier to the cytokine storm in elderly coronavirusinfected people, thus reducing the risk of severe disease progression and death. Conceptualization -KK; Investigation -JMP; Methodology -KK, Writing-original draft -JMP, KK; Writingreview & editing -JMP, KK. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Immunosenescence: emerging challenges for an ageing population SARS-CoV-2 may regulate cellular responses through depletion of specific host miRNAs Distribution of SARS-CoV-2 PCR cycle threshold values provide practical insight into overall and target-specific sensitivity among symptomatic patients Association of the CT values of realtime PCR of viral upper respiratory tract infection with clinical severity Age-related differences in nasopharyngeal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels in patients with mild to moderate coronavirus disease 2019 (COVID-19) Senolytics decrease senescent cells in humans How COVID-19 induces cytokine storm with high mortality Age-related morbidity and mortality among patients with COVID-19 Relationship of the cycle threshold values of SARS-CoV-2 polymerase chain reaction and total severity score of computerized tomography in patients with COVID 19 Interleukin-6 in aging and chronic disease: a magnificent pathway Laboratory diagnosis of SARS-CoV-2 -a review of current methods Causes, consequences, and reversal of immune system aging Activation of innate immunity system during aging: NF-kB signaling is the molecular culprit of inflamm-aging Cytokine storm in COVID-19: the current evidence and treatment strategies Laboratory diagnosis of COVID-19: current issues and challenges To interpret the SARS-CoV-2 test, consider the cycle threshold value An update on inflamm-aging: mechanisms, prevention, and treatment COVID-19 diagnosis -a review of current methods Authors of the study warmly thank all diagnosticians, molecular biologists, as well as technical and administrative staff working on SARS-CoV-2 diagnostics at Poznan University of Medical Sciences Coronavirus Laboratory for their daily hard work and dedication.