key: cord-0968568-v1p5zmlu
authors: Cuevas-Ferrando, E.; Randazzo, W.; Perez-Cataluna, A.; Falco, I.; Navarro, D.; Martin-Latin, S.; Diaz-Reolid, A.; Giron-Guzman, I.; Allende, A.; Sanchez, G.
title: Viability RT-PCR for SARS-CoV-2: a step forward to solve the infectivity quandary
date: 2021-03-26
journal: nan
DOI: 10.1101/2021.03.22.21253818
sha: 36a33f0a528d02ad1f1fb235b26c80a902ff8754
doc_id: 968568
cord_uid: v1p5zmlu

Background: Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. Methods: We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. Findings: We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. Interpretation: The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum. Fundings: This work was supported by Spanish Scientific Research Council (CSIC), Generalitat Valenciana, and MICINN co-founded by AEI/FEDER, UE.

Introduction monoazide (EMA™, Geniul, Spain) was diluted in dimethylsulfoxide (DMSO) to 2·0 mM, PEMAX™ 93 (Geniul, Spain) and propidium monoazide (PMAxx™, Biotium, CA, US) were diluted in nuclease-94 free water to 4·0 mM, platinum (IV) chloride (PtCl4; Acros Organics, NJ, US) and cis-95 diamineplatinum (II) dichloride (CDDP; Sigma-Aldrich, MO, US) salts were dissolved in DMSO to 96 1·0 M and further diluted in nuclease-free water to 50 mM. Viability assays were carried out by 97 treating 300 µL of either genomic SARS-CoV-2 RNA (approx. 10 3 gc/mL), gamma-inactivated 98 (approx. 10 5 gc/mL), and heat inactivated SARS-CoV-2 (approx. 10 5 gc/mL) suspensions with final 99

concentrations of 50-100 µM photoactivatable dyes (PMAxx™, PEMAX™, or EMA™) or 0·1-2·0 100 mM platinum compounds (CDDP or PtCl4) in DNA LoBind tubes (Eppendorf, Germany). 101

Photoactivation of monoazide dyes was achieved by 10 min of dark-incubation in an orbital 102

shaker (150 rpm) at room temperature (RT) followed by 15 min blue LED light exposure in a 103 photo-activation system (Led-Active Blue, GenIUL). Alternatively, 30 min incubation at RT in an 104 orbital shaker (150 rpm) were used for viability treatments with platinum compounds. A control 105 consisting of genomic RNA or virus suspension without viability marker was included in each 106 assay. Following the viability treatment, the viral RNA was immediately purified as described 107

hereafter. 108

contaminated samples 110

Platinum (IV) chloride was selected as the most reliable viability marker and tested at final 111 concentrations of 0·5 to 5·0 mM for viability RT-qPCR optimization in stool, urine, 112 nasopharyngeal swabs and wastewater samples. 113

For the initial optimization, stool and urine specimens that had tested negative for SARS-CoV-2 114

were retrieved from IATA biobank. Faecal material was resuspended 1% w/v in phosphate-115 buffered saline (PBS), and supernatant recovered by centrifugation at 2000 × g for 5 min. Direct 116 and ten-fold diluted urine, and ten-fold diluted faecal suspension (final 1% w/v faecal dilution) 117

were spiked with either gamma-and/or heat inactivated SARS-CoV-2 to approximately 10 5 gc/L 118 final concentration. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; dilutions were consistently tested to check RT-qPCR inhibition due to viability marker residues 145 or inhibitory substances in the sample. 146

Statistical analysis 147

All data were compiled from three independent experiments with at least two technical 148

replicates for each variable. Data are presented as median ± SD. Significant differences in median 149 cycle threshold (Ct) were determined by using either one-or two way(s) ANOVA followed by 150

Dunnett's multiple comparisons test on GraphPad Prism version 8·02 (GraphPad Software, US). 151

Differences in means were considered significant when the p was <0·05. 152

Results 153

Initial assessment of viability markers and RT-qPCR assays 154

With regard to the viability markers tested, platinum compounds were better at preventing PCR 155 amplification of SARS-CoV-2 genomic RNA suspension than monoazide dyes, regardless of the 156 RT-qPCR target ( Figure 1 ). while the superior ability of PtCl4 was confirmed for both gamma-and heat inactivated SARS-170

CoV-2 viral particles ( Figure 2) . A final concentration of 1·0 mM PtCl4 was needed to consistently 171 prevent the amplification of inactivated viruses by viability RT-qPCR. Thus, we further applied 172 the PtCl4 viability RT-qPCR to high concentrated gamma-inactivated viral suspensions (ca. 8·50 x 173 10 6 gc/mL). Results showed that 0·5 and 1·0 mM PtCl4 reduced by 3·4 and 6·8 Cts compared to 174 the control. Although significant statistical differences were detected for all treatments 175 regardless of the concentration of the metal compound, only 2·0 mM PtCl4 showed to 176 consistently prevent signal amplification (only one positive out of 8 replicates, Ct=39·11). 177

Effect of sample complexity on viability RT-qPCR 178

To determine the effect of sample matrix on viability RT-qPCR, we spiked 10-fold diluted stool 179

suspensions (1% w/v final dilution) and urine specimen (10% v/v final dilution) with 180 approximately 10 5 gc/mL gamma-inactivated SARS-CoV-2, and applied up to 5·0 mM PtCl4 as 181 viability marker. Compared to the untreated control, significant differences were observed for 182 1·0 mM PtCl4 in urine samples or 1·25 mM PtCl4 in stool suspensions ( Figure 3 ). However, a 183 concentration of 5·0 mM was needed to completely remove the PCR signals in urine, while 2·5 184 mM PtCl4 prevented the amplification of 1 out of 8 replicates in stool. Although the complete 185 inhibition of amplification signals was achieved to a limited extent, a sharp difference above one 186 logarithm of genomic copies (ΔCts≈3·3) was observed in stool and urine samples processed with 187

1·25 and 3·75 mM PtCl4, respectively. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint demonstrated that viability RT-qPCR efficiently discriminated free RNAs and inactivated SARS-220

CoV-2 inoculated in buffer, stool and urine suspensions. Then, we further proved that the 221 method inferred SARS-CoV-2 infectivity better than RT-qPCR alone in both nasopharyngeal 222 swabs from positive COVID-19 patients and in naturally contaminated wastewater samples. In 223 the case of complex matrices, increased PtCl4 concentration of 2·5 mM and ten-fold sample 224 dilution are recommended because of the presence of suspended solids and inhibitors that 225 hinder the efficacy of the viability treatment. 226

Our investigation initially included five well-established molecular assays since the length of the 227 amplicon and/or the richness of secondary structures of targeted RNA may affect the efficiency 228 of viability treatments. (17, 18) We show that metal compounds performed better than monoazide 229 dyes, irrespective of RT-qPCR assays. However, RT-qPCR targeting N1 region is recommended 230 because of its superior sensitiveness. This aspect is of importance because complex matrices 231 needed to be diluted to achieve a more efficient inference of viral infectivity, as demonstrated 232

in spiked stool and urine, in positive swabs and in contaminated wastewater. Similarly, sample 233 dilution was needed to implement a viability RT-qPCR targeting norovirus in sewage. (19)  234 Moreover, N1 assay better fits the testing on samples with expected low viral concentrations 235 (e.g., environmental samples) and/or PCR inhibitors (e.g., concentrated wastewater, stool). As 236 N1 assay has been validated in many laboratories worldwide, this viability method could also be 237 easily and widely implemented. 238

To the best of our knowledge, this is the first report on a rapid molecular assay independent 239 from viral replication in cell culture developed to test SARS-CoV-2 infectivity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint worth to report that we also tested on inactivated SARS-CoV-2 suspensions a porcine gastric 249 mucine in situ capture RT-qPCR, a method that was originally implemented in our laboratory for 250 human enteric viruses. (20) Although proper SARS-CoV-2 infectious controls could not be included, 251 those experiments resulted in inconclusive outcomes (data not shown). 252

Our viability RT-qPCR results of nasopharyngeal swabs from positive COVID-19 patients indicate 253 the potential infectivity of the samples, while naturally contaminated wastewater are unlikely 254 to contain infectious viral particles. These later findings reflect the viral replication in cell culture 255 from RNA positive stool and respiratory samples as well as the unsuccessful attempts to isolate 256 and cultivate SARS-CoV-2 from wastewater samples. (21-23) 257

Our findings are clinically relevant as RT-qPCR has become the primary method to diagnose 258 COVID-19. However, as it detects RNA, its ability to determine the infectivity of patients is 259

limited. ( it could not be retrieved as de-identified specimens were analysed in this study. 278

Despite the viability treatment, we detected residual signals in heat inactivated nasopharyngeal 279

swabs. This could be attributed to the viral envelop and nucleoproteins that limit the access 280 and/or the binding of viability markers to SARS-CoV-2 RNA, as hypothesised for avian influenza 281 virus and bacteriophage T4. (12, 29) The enveloped structure of coronaviruses may also explain the 282 increased concentration of viability markers needed for SARS-CoV-2 and PEDV compared to 283 human enteric viruses. (13, 19, 30) This cumbersome finding obtained by the proposed viability 284 procedure suggests that the overestimation of the infectivity of a given sample may occur which, 285

although warranting a careful interpretation, represents a conservative prediction. With regards 286

to wastewater samples that tested positive for SARS-CoV-2 RNA, they most probably contained 287 detergents and chemicals that are detrimental to viral infectivity further supporting the efficacy 288 of the viability RT-qPCR in discriminating potentially infectious and inactivated viral particles. (31) 289

The ultimate confirmation on the infectivity of the samples by cell culture, although 290 recommendable, could not be provided. Detection of SARS-CoV-2 by either culture and viability 291

RT-qPCR is valuable as a proxy for infectiousness; however, as the human infectious dose 292 remains unknown, the significance of low titres of infectious virus for human-to-human 293 transmission remains uncertain. Above all, as some individuals reportedly remain PCR positive 294 weeks after SARS-CoV-2 infection recovery, knowing whether viral RNA in these persistent 295

carriers is contagious provides key insights for quarantine policy, to safely discontinue self-296 isolation and contact tracing as essential public health measures to definitively prevent 297 transmission. (6,26,32) Besides some limitations, the proposed viability RT-qPCR effectively reduced 298 the amplification signals of non-infectious and free RNA of SARS-CoV-2 in complex matrices 299 finally providing a better estimation of the infectiousness of samples. Thus, mathematical 300 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; models derived from laboratory scale experiments comparing viability RT-qPCR and viral 301 replication could correlate viral load and infectivity, finally providing relevant tools of interest 302 based on rapid molecular assay for prevention strategies and risk assessment. 303

Conclusions 304

In conclusion, the use of pre-treatments to prevent RT-qPCR amplification of RNA from non-305

infectious SARS-CoV-2 using platinum chloride as a viability marker of infectivity was 306 implemented in stool and urine samples and successfully validated in naturally contaminated 307 wastewater samples, supporting the idea that SARS-CoV-2 present in sewage is not infectious. 308

Residual amplification signals in nasopharyngeal swabs exposed to heat-inactivation 309 overestimated the amount of viable virus, still providing a conservative interpretation of the 310 infectiousness of the sample. Our study proposes a rapid analytical tool based on viability RT-311 qPCR to infer SARS-CoV-2 infectivity with potential application in risk assessment, and 312 prevention and control in public health programmes. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.22.21253818 doi: medRxiv preprint

Droplets and aerosols in the transmission of SARS-CoV-2

Potential intestinal infection and faecal-oral

Antiviral 383 activity of aged green tea extract in model food systems and under gastric conditions

Presence 386 and infectivity of SARS-CoV-2 virus in wastewaters and rivers

Virological 389 assessment of hospitalized patients with COVID-2019

Duration of 391 infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19

Prolonged detection of Severe Acute Respiratory 394 Syndrome Coronavirus 2 (SARS-CoV-2) RNA in an obstetric patient with antibody 395 seroconversion

Prolonged virus shedding 397 even after seroconversion in a patient with COVID-19

SARS-CoV-2 399 persistence is associated with antigen-specific CD8 T-cell responses

Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 404 (COVID-19) in the United States

Discrimination of 406 infectious bacteriophage T4 virus by propidium monoazide real-time PCR

Viability RT-qPCR to distinguish 409 between HEV and HAV with intact and altered capsids

Viricidal treatments for prevention of 411 coronavirus infection

How should a positive PCR test result 413 for COVID-19 in an asymptomatic individual be interpreted and managed?

Added value of this study 463 We show that SARS-CoV-2 RNA amplification of non-infectious samples can be reproducibly 464 prevented by processing clinical and wastewater samples by a platinum chloride viability RT-465 qPCR technique. To the best of our knowledge this is the first report on this subject that also 466 provides the following distinguishing features: (i) the procedure was optimized using standard 467 materials which include SARS-CoV-2 genomic RNA, gamma-and heat inactivated viral particles; 468(ii) the preliminary findings were validated in nasopharyngeal swabs from COVID-19 positive 469 patients and in wastewater samples naturally contaminated with SARS-CoV-2 RNA. 470Overall, this study allows to selectively detect SARS-CoV-2 RNA belonging to potentially 471 infectious viral particles in complex matrices of interest for epidemiological surveillance and 472 pandemic control. 473Implications of all the available evidence 474Extensive lockdown measures are currently allowing to partially mitigate the spread of SARS-475CoV-2. As such, it is of extreme importance to set up feasible and reliable epidemiological 476 surveillance strategies that include clinical and environmental monitoring programs. 477Information on the infectivity of such samples is the keystone to better define the transmission 478 route(s) and the risk of exposure. Further developments of predictive models based on such a 479 viability approach may have a broad range of applications: including risk assessment, food safety 480 and public health policy. 481