tetano
Editor, Senior Moderator
Microbiol Spectr
. 2024 Jul 22:e0016024.
doi: 10.1128/spectrum.00160-24. Online ahead of print. Advancing COVID-19 diagnostics: rapid detection of intact SARS-CoV-2 using viability RT-PCR assay
Judith M J Veugen[SUP] 1 2 3 4 [/SUP], Tom Schoenmakers[SUP] 5 6 [/SUP], Inge H M van Loo[SUP] 3 4 [/SUP], Bart L Haagmans[SUP] 7 [/SUP], Mathie P G Leers[SUP] 5 8 [/SUP], Mart M Lamers[SUP] 7 [/SUP], Mayk Lucchesi[SUP] 3 4 [/SUP], Bas C T van Bussel[SUP] 4 9 10 [/SUP], Walther N K A van Mook[SUP] 9 11 [/SUP], Rudy M M A Nuijts[SUP] 1 2 12 [/SUP], Paul H M Savelkoul[SUP] 3 [/SUP], Mor M Dickman[SUP] 1 2 [/SUP], Petra F G Wolffs[SUP] 3 4 [/SUP]
Affiliations
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.
Keywords: PMAxx; SARS-CoV-2; infectiousness; intact virus particles; viability PCR; viral shedding.
. 2024 Jul 22:e0016024.
doi: 10.1128/spectrum.00160-24. Online ahead of print. Advancing COVID-19 diagnostics: rapid detection of intact SARS-CoV-2 using viability RT-PCR assay
Judith M J Veugen[SUP] 1 2 3 4 [/SUP], Tom Schoenmakers[SUP] 5 6 [/SUP], Inge H M van Loo[SUP] 3 4 [/SUP], Bart L Haagmans[SUP] 7 [/SUP], Mathie P G Leers[SUP] 5 8 [/SUP], Mart M Lamers[SUP] 7 [/SUP], Mayk Lucchesi[SUP] 3 4 [/SUP], Bas C T van Bussel[SUP] 4 9 10 [/SUP], Walther N K A van Mook[SUP] 9 11 [/SUP], Rudy M M A Nuijts[SUP] 1 2 12 [/SUP], Paul H M Savelkoul[SUP] 3 [/SUP], Mor M Dickman[SUP] 1 2 [/SUP], Petra F G Wolffs[SUP] 3 4 [/SUP]
Affiliations
- PMID: 39037224
- DOI: 10.1128/spectrum.00160-24
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.
Keywords: PMAxx; SARS-CoV-2; infectiousness; intact virus particles; viability PCR; viral shedding.