Advancing COVID-19 diagnostics: rapid detection of intact SARS-CoV-2 using viability RT-PCR assay

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness. IMPORTANCE This study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

1st Editorial Decision Re: Spectrum00160-24 (Advancing COVID-19 diagnostics: Rapid detection of intact SARS-CoV-2 using viability RT-PCR assay) Dear Dr. Petra F.G. Wolffs: Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
Please note comments from Reviewer 1 in particular.Additional verification of the assay with the appropriate controls should be considered.
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Sincerely, Anne Wyllie Editor Microbiology Spectrum
Reviewer #1 (Comments for the Author): To the test tubes, PMA and SDS were added and the tubes were incubated for 30 minutes at 37 degrees and subsequently subjected to photolysis, and then lysis buffer was added.The control tubes were not incubated, did not receive SDS and were not subjected to photolysis but were lysed immediately.The possible influence of the 30 minutes incubation at 37, the SDS, or high-energy blue light, are thus not taken into account.An additional control in the form of extracted virus RNA treated with PMA and without PMA would have been appropriate to demonstrate the actual function of the PMA.
It is not clear to what extent the viral RNA becomes exposed by the heat treatment.Inactivation of the virus is not proven by plaque analysis.
Reviewer #2 (Comments for the Author): Authors have tried a novel assays for SARS COVID .Experiment is well designed and done comprehensively, however there is a need for little more discussion on analytical sensitivity and specificity of the assay and also it's diagnostic sensitivity and specificity in comparison to the gold standard assay like Real Time PCR .This can be added as a limitation of the study also.

Response to reviewer comments
Editor Please note comments from Reviewer 1 in particular.Additional verification of the assay with the appropriate controls should be considered.
We thank the editor for the opportunity to revise our paper.We performed an additional verification with the appropriate controls as suggested.The details of the additional verification can be found in our response to reviewer #1, point 1 section.
Reviewer #1 1.To the test tubes, PMA and SDS were added and the tubes were incubated for 30 minutes at 37 degrees and subsequently subjected to photolysis, and then lysis buffer was added.The control tubes were not incubated, did not receive SDS and were not subjected to photolysis but were lysed immediately.The possible influence of the 30 minutes incubation at 37, the SDS, or high-energy blue light, are thus not taken into account.An additional control in the form of extracted virus RNA treated with PMA and without PMA would have been appropriate to demonstrate the actual function of the PMA.
Response: We thank the reviewer for this suggestion, and we agree that additional experiments to demonstrate the actual function of PMAxx and the potential influence of the 30 min incubation at 37˚C, 0.005% SDS, and the PMA lite is valuable.In response to this suggestion, we conducted additional experiments in duplicate using extracted SARS-CoV-2 RNA.The results obtained clearly indicate that the 30 min incubation at 37˚C, 0.005% SDS, and the PMA lite alone do not affect the v-PCR assay.We only observed an effect when these steps were combined with the pretreatment of samples using PMAxx (Table 1).The following paragraphs were added to the manuscript: "To evaluate the potential impact of the 30-minute incubation at 37°C, 0.005% SDS, and the PMA lite, experiments were performed using extracted SARS-CoV-2 RNA.The v-PCR assay was performed for each step separately to assess its individual influence."(lines 138-140, materials and methods) and

"Functionality PMAxx
To demonstrate the functionality of PMAxx and assess the potential influence of the 30-minute incubation at 37˚C, 0.005% SDS, and the PMA lite, duplicated experiments were performed using extracted SARS-CoV-2 RNA.The individual steps of 30-minute incubation at 37˚C, 0.005% SDS, and the PMA lite did not affect the v-PCR assay, as evidenced by mean delta Ct values of 0.4, 0.4, and 0.5 respectively.However, an effect was observed when these steps were combined with the pretreatment using PMAxx, resulting in a mean delta Ct value of >-13.5, corresponding to a mean log reduction >4.1." (lines 188-195, results) 2. It is not clear to what extent the viral RNA becomes exposed by the heat treatment.Inactivation of the virus is not proven by plaque analysis.
Response: We appreciate the reviewer raising this important point regarding verification of SARS-CoV-2 inactivation.As the reviewer correctly noted, we were unable to prove complete inactivation of the virus as no culturing after inactivation was performed.
During optimization of the protocol, we did attempt multiple heat inactivation protocols, including heating at 60°C for various time durations.However, we found these were insufficient as the maximum delta Ct value achieved was only -0.9.Upon heat inactivation for 5 min at 95˚C, we were able to reach a maximum delta Ct value of -14.2, similar to the inactivation of naked RNA.While we assumed successful inactivation, we acknowledge we could not definitively prove this without culturing experiments and added this as a limitation to the manuscript : "While successful inactivation was assumed, definitive proof would require culturing experiments, which were not performed."(lines 244-245, discussion) Reviewer #2 1. Authors have tried a novel assays for SARS COVID .Experiment is well designed and done comprehensively, however there is a need for little more discussion on analytical sensitivity and specificity of the assay and also it's diagnostic sensitivity and specificity in comparison to the gold standard assay like Real Time PCR.
Response: We acknowledge the reviewer's comment regarding the need for a more comprehensive discussion on the analytical sensitivity and specificity of the v-PCR in comparison to the gold standard assay.
The sensitivity and specificity of the v-PCR are influenced by the choice of the PCR used after pMAXX treatment.The method can be combined with any PCR, provided the fragment is long enough to achieve good pMAXX binding.
Regarding the sensitivity of the current v-PCR setup, we found it to be approximately 5 copies/PCR.
To evaluate the specificity of the v-PCR, we applied the v-PCR in a large clinical study and compared the results of the v-PCR with our current routine clinical diagnostics PCR.During this comparison, no false positive results were observed with the v-PCR We added the following text to the discussion: "Importantly, this technique can be combined with any PCR assay while preserving the characteristics and performance of the original assay, provided the fragment is long enough for optimal pMAXX binding."(lines 251-253, discussion) June 10, 2024 1st Revision -Editorial Decision Re: Spectrum00160-24R1 (Advancing COVID-19 diagnostics: Rapid detection of intact SARS-CoV-2 using viability RT-PCR assay) Dear Dr. Petra F.G. Wolffs: Thank you for your thoughtful edits in response to the reviewer's comments.
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Table 1 .
Cycle threshold (Ct) values and delta Ct (ΔCt) values of SARS-COV-2 RNA to demonstrate the actual function of PMAxx.ΔCt values are calculated by subtracting the Ct value of the treated sample from the direct lysed sample.UD, undetectable: no detectable Ct value that exceeded the threshold within 42 cycles.