key: cord-0996376-urqdvkrl
authors: Zurita-Cruz, Nelly Daniela; Martín-Ramírez, Alexandra; Rodríguez-Serrano, Diego Aníbal; González-Álvaro, Isidoro; Roy-Vallejo, Emilia; la Cámara, Rafael De; García-Rodrigo, Leticia Fontán; Domingo, Laura Cardeñoso
title: Usefulness of real-time RT-PCR to understand the kinetics of SARS-CoV-2 in blood: a prospective study
date: 2022-04-26
journal: J Clin Virol
DOI: 10.1016/j.jcv.2022.105166
sha: 2c32865d6ccddbf30fef707000c6f2f2018f3625
doc_id: 996376
cord_uid: urqdvkrl

BACKGROUND: : SARS-CoV-2 viral load and kinetics assessed in serial blood samples from hospitalised COVID-19 patients by RT-PCR are poorly understood. METHODS: : We conducted an observational, prospective case series study in hospitalised COVID-19 patients. Clinical outcome data (Intensive Care Unit admission and mortality) were collected from all patients until discharge. Viremia was determined longitudinally during hospitalisation, in plasma and serum samples collected sequentially, using two commercial and standardised RT-PCR techniques approved for use in diagnosis of SARS-CoV-2. Viral load (copies/mL and log10) was determined with quantitative TaqPath™COVID-19 test. Persistent viremia (PV) was defined as two or more consecutive quantifiable viral loads detected in blood samples (plasma/serum) during hospitalisation. RESULTS: : SARS-CoV-2 viremia was studied in 57 hospitalised COVID-19 patients. PV was detected in 16 (28%) patients. All of them, except for one who rapidly progressed to death, cleared viremia during hospitalisation. Poor clinical outcome occurred in 62.5% of patients with PV, while none of the negative patients or those with sporadic viremia presented this outcome (p<0.0001). Viral load was significantly higher in patients with PV than in those with Sporadic Viremia (p<0.05). Patients presented PV for a short period of time: median time from admission was 5 days (Range=2-12) and 4.5 days (Range=2-8) for plasma and serum samples, respectively. Similar results were obtained with all RT-PCR assays for both types of samples. CONCLUSIONS: : Detection of persistent SARS-CoV-2 viremia, by real time RT-PCR, expressed as viral load over time, could allow identifying hospitalised COVID-19 patients at risk of poor clinical outcome.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first described in 65

December 2019 in Wuhan, China(1). As of 19th of January 2022, 5,542,359 deaths were 66 reported to WHO(2) . 67

While most COVID-19 patients present mild disease, others develop a severe disease(3). 68

Identifying patients at risk of developing severe COVID-19 is an unmet need to improve the 69 management of these patients and prevent morbidity and mortality. Some risk factors of poor 70 prognosis, such as elevated concentrations of interleukin (IL)-6, contribute to risk stratification 71 of COVID-19 patients (4-6) 72

Detection of SARS-CoV-2 RNA by real-time reverse transcription polymerase chain reaction 73 (RT-PCR) in nasopharyngeal swabs is the gold standard for COVID-19 diagnosis(7). However, 74 no consistent association has been reported between the viral load in these samples and disease 75 7 Biosystems, Whaltman, MA, USA) and according to Thermocycler manufacturer 137 specifications(15). Detailed description of standard curve determination is shown in 138 supplementary material. Samples were considered quantifiable when mean Ct in the duplicate 139 test for each gene was ≤37 and standard deviation (SD) was <0.5. All results not fulfilling these 140 criteria and/or those with detection in only one duplicate, were considered positive, but not 141 quantifiable. To estimate a value for quantification limit, median of all non-quantifiable values 142 was calculated, obtaining 17.75 copies/mL for serum samples and 14.89 copies/mL for plasma 143 samples. For the genes that met quantification criteria, mean quantity was calculated from the 144 duplicates, expressed as copies/mL and logarithm with base 10(log 10 ). Two positive controls 145 (corresponding to 20,000 and 200 copies) and two negative controls were added in each run in 146 duplicate. 147

Moreover, we studied the number of genes (N, S or ORF) that were amplified in each plasma 148 sample tested by q-RT-PCR. 149 150

The presence of SARS-CoV-2 RNA was detected by qualitative RT-PCR methods and viral 152 load was determined by qTaqPath-test in plasma and serum of each patient collected throughout 153 the follow-up. To analyse viremia kinetics, time course curves were obtained plotting viral load 154 change over time. Specific patterns of viral load change were identified and their relationship 155 with clinical evolution was analysed. 156

Three main outcomes in the considered study were: in-hospital all-cause mortality, Intensive 158

Care Unit (ICU) admission and the combination of both (Poor outcome). Persistent Viremia 159

(PV) was defined as SARS-CoV-2 RNA detected in two or more consecutive determinations. 160 

Fifty-seven patients consecutively admitted to our hospital were included in this study ( Figure  172 1 showed that at least two assay targets could be detected in 79% of the patients: all 3 targets were 225 detected in 64.3%, 2 targets in 14,3% and only the N gene in 21.4% of the cases. (Figure 4) . whereas for PV median viral load was 558 copies/mL (IQR= 170.3-1145.4 ; mean= 2.71±0.61 292 log 10 ) for plasma and 370.4 copies/mL (IQR= 180.92-1233.6 ; mean=2.57±0.55 log 10 ) for serum. 293 Viral load was significantly higher for PV compared to SV for both types of samples (Figure 8) . 294 295 Figure 8 . Viral load is significantly higher in patients with persistent viremia than those with 296 sporadic viremia in serum and plasma samples. The viral load expressed as log 10 showed a 297 normal distribution, so the difference of viral loads between both groups was analysed by 298

The hypothesis of this study was that detectable SARS-CoV-2 viremia in successive samples 301 over time (persistent viremia) could identify hospitalised COVID-19 patients with high risk of 302 poor clinical outcome. To verify this, we studied methods for qualitative detection of SARS-303

CoV-2 RNA and for quantification of viral load in plasma and serum, using commercially 304 available RT-PCR kits, marked with CE and FDA authorisation. In addition, the association of 305 viremia evolution with patient outcome was also evaluated. 306

Regarding the relationship between disease severity and the pattern of viremia kinetics, our 307 results showed that all patients with ICU admission and/or death during hospitalisation had PV, 308

while none of the patients with SV or NV had poor clinical outcome in this cohort. This 309 supports our hypothesis that patients with PV are more likely to have a poor clinical outcome, in 310 agreement with other authors. (17-23) . 311

The graphical representation of viremia kinetics patterns in patients with PV, assessed by two 312 qualitative and one quantitative RT-PCR methods showed similar kinetic patterns regardless of 313 the use of different reagents and RT-PCR platforms. This analysis suggests that the dynamics of 314 viremia in longitudinal samples correlates with the presence of SARS-CoV-2 RNA in blood and 315 is reproducible. However, some discrepancy values were detected in isolated cases where the Ct 316 value or viral load was lower than those in the paired sample from the same blood extraction 317 (plasma vs. serum). We hypothesised that those discrepancies could be due to a loss of sample 318 integrity(24). However, the fact that different techniques can be used to analyse the kinetics of 319 the SARS-CoV-2 viremia increases the versatility of this determination when it comes to its 320 implementation in clinical microbiology laboratories. 321

Our previous results suggested that the Ct value is a good approximation for the stratification of 322 severely ill patients(12). However, the variation due to the sample type and handling, the 323 technique used, and the intrinsic variability of RT-PCR makes it necessary to develop a 324 standardised method to obtain more reproducible results(25). Even in this series, both assays 325 detected a higher number of positives in plasma than in serum. In this sense, it is well known 326 that the quantification of viral load in plasma has been standardised in other pathologies 327 associated to viral infections such as HIV, HCV, HBV, , and provides a more 328 precise and reliable monitoring of viremia than Ct values. For this reason, we propose the use of 329 SARS-CoV-2 viral load from plasma samples for the analysis of viremia kinetics. 330

Different authors have quantified viremia using new technologies such as Droplet Digital PCR 331 (18, 30)using internally developed methods with primer design(17), or ultrasensitive 332 quantitative . In this context, the objective of this work was to assess the usefulness 333 of a quantification method using RT-PCR to monitor SARS-CoV-2 viral load in hospitalised 334 COVID-19 patients. We used commercial and standardised reagents with CE and FDA marking 335 for authorisation, making a standard curve and taking advantage of the options offered by the 336

QuantStudio 5 thermal cycler interpretation software. Taken all together, these characteristics 337 provide a robust and reproducible quantification method that allows monitoring viremia in 338 hospitalised COVID-19 patients. Also, we agree with other authors on the need to develop 339 standardised techniques for quantifying viral load in blood, with FDA or CE approval, that 340 could contribute to improve the management of patients with 21) . 341

Some authors have referred to the detection of SARS-CoV-2 RNA in blood as RNAemia(13, 342 24, 30) . However, we use the expression "viremia" for this determination, as the presence of 343 viral RNA in the blood, in line with other published studies(17, 23, 31-33), since in 344 approximately 80% of the studied plasma samples 2 or 3 SARS-CoV-2 genes were detected. 345

Overall results of single viremia determinations within the first week do not allow analysing the 346 association with clinical outcome whereas persistent viremia could be associated with Poor 347

Outcome. This preliminary result suggests that this variable, which reflects early viral load 348 kinetics, may be suitable for assessment of outcome in clinical practice. Nevertheless, this 349 20 possibility should be further analysed in studies with higher sample size and using a more 350 detailed experimental approach, which are beyond the scope of this study. 351

The present study has certain limitations. First, the number of patients included in the study was 352 moderate (57 patients). Second, although two RT-PCR techniques were performed, only the 353 TapPath™ test allowed us to quantify the viral load (in copies/ml and log10 viral load). Third, 354 prolonged hospitalization may increase the number of viremia determinations and therefore the 355 likelihood of detecting persistent viremia. However, persistent viremia was detected within the 356 first week of hospitalisation in our cohort thereby strongly suggesting that prolonged 357 hospitalisation could not cause this bias in our results. 358

In summary, we conclude that persistent SARS-CoV-2 viremia in blood samples may be 

The authors declare that they have no known competing financial interests or personal 377 relationships that could have appeared to influence the work reported in this paper. 378

This research did not receive any specific grant from funding agencies in the public, 380 commercial, or not-for-profit sectors. 381

The work of E. Roy-Vallejo has been funded by a Rio-Hortega grant CM19/00149 from the 382 Ministerio de Economía y Competitividad (Instituto de Salud Carlos III) and co-funded by The 383

European Regional Development Fund (ERDF) "A way to make Europe" 384 385 386

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