Determining the impact of vaccination on SARS-CoV-2 RT-PCR cycle threshold values and infectious viral titers

ABSTRACT Background: As the COVID-19 pandemic continues, efforts to better understand SARS-CoV-2 viral shedding and transmission in both unvaccinated and vaccinated populations remain critical to informing public health policies and vaccine development. The utility of using RT-PCR cycle threshold values (CT values) as a proxy for infectious viral titers from individuals infected with SARS-CoV-2 is yet to be fully understood. This retrospective observational cohort study compares quantitative infectious viral titers derived from a focus-forming viral titer assay with SARS-CoV-2 RT-PCR CT values in both unvaccinated and vaccinated individuals infected with the Delta strain. Methods: Nasopharyngeal swabs positive for SARS-CoV-2 by RT-PCR with a CT value < 27 collected from June 26th to October 17th of 2021 at the University of Vermont Medical Center Clinical Laboratory for which vaccination records were available were included. Partially vaccinated and individuals < 18 years of age were excluded. Infectious viral titers were determined using a micro-focus forming assay under BSL-3 containment. Results: 119 specimens from 22 unvaccinated and 97 vaccinated individuals met all inclusion criteria and had sufficient residual volume to undergo viral titering. A negative correlation between RT-PCR CT values and viral titers was observed in both unvaccinated and vaccinated groups. No difference in mean CT value or viral titer was detected between vaccinated and unvaccinated groups. Viral titers did not change as a function of time since vaccination. Conclusions: Our results add to the growing body of knowledge regarding the correlation of SARS-CoV-2 RNA levels and levels of infectious virus. At similar CT values, vaccination does not appear to impact an individual's potential infectivity.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age

At the start of the SARS-CoV-2 pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, however they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in pediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virologic features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children infected with COVID-19, we compared viral RNA levels (clinical RT-qPCR CTs) and infectious virus titers from 144 SARS-CoV-2 positive clinical samples collected from children ages 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age

At the start of the SARS-CoV-2 pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, however they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in pediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virologic features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children infected with COVID-19, we compared viral RNA levels (clinical RT-qPCR CTs) and infectious virus titers from 144 SARS-CoV-2 positive clinical samples collected from children ages 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age

At the start of the SARS-CoV-2 pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, however they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in pediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virologic features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children infected with COVID-19, we compared viral RNA levels (clinical RT-qPCR CTs) and infectious virus titers from 144 SARS-CoV-2 positive clinical samples collected from children ages 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

All Hands-On Deck and All Decks on Hand: Surmounting Supply Chain Limitations During the COVID-19 Pandemic.

Testing during the COVID-19 pandemic has been crucial to public health surveillance and clinical care. Supply chain constraints-spanning limitations in testing kits, reagents, pipet tips, and swabs availability-have challenged the ability to scale COVID-19 testing. During the early months, sample collection kits shortages constrained planned testing expansions. In response, the University of Vermont Medical Center, University of Vermont College of Medicine, Vermont Department of Health Laboratory, Aspenti Health, and providers across Vermont including 16 area hospitals partnered to surmount these barriers. The primary objectives were to increase supply availability and manage utilization. Within the first month of Vermont's stay-at-home order, the University of Vermont Medical Center laboratory partnered with College of Medicine to create in-house collection kits, producing 5000 per week. University of Vermont Medical Center reassigned 4 phlebotomists, laboratory educators, and other laboratory staff, who had reduced workloads, to participate (requiring a total of 5.3-7.6 full-time equivalent (FTE) during the period of study). By August, automation at a local commercial laboratory produced 22,000 vials of media in one week (reducing the required personnel by 1.2 FTE). A multisite, cross-institutional approach was used to manage specimen collection kit utilization across Vermont. Hospital laboratory directors, managers, and providers agreed to order only as needed to avoid supply stockpiles and supported operational constraints through ongoing validations and kit assembly. Throughout this pandemic, Vermont has ranked highly in number of tests per million people, demonstrating the value of local collaboration to surmount obstacles during disease outbreaks and the importance of creative allocation of resources to address statewide needs.

Kinetics and isotype assessment of antibodies targeting the spike protein receptor-binding domain of severe acute respiratory syndrome-coronavirus-2 in COVID-19 patients as a function of age, biological sex and disease severity.

OBJECTIVES: There is an incomplete understanding of the host humoral immune response to severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2, which underlies COVID-19, during acute infection. Host factors such as age and sex as well as the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor-binding domain of the CoV spike (RBD-S) protein mediates host cell binding and infection and is a major target for vaccine design to elicit neutralising antibodies. METHODS: We assessed serum anti-SARS-CoV-2 RBD-S IgG, IgM and IgA antibodies by a two-step ELISA and neutralising antibodies in a cross-sectional study of hospitalised COVID-19 patients of varying disease severities. Anti-RBD-S IgG levels were also determined in asymptomatic seropositives. RESULTS: We found equivalent levels of anti-RBD-S antibodies in male and female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti-RBD-S IgG, IgM and IgA responses were simultaneously induced within 10 days after onset, with anti-RBD-S IgG sustained over a 5-week period. Anti-RBD-S antibodies strongly correlated with neutralising activity. Lastly, anti-RBD-S IgG responses were higher in symptomatic COVID-19 patients during acute infection compared with asymptomatic seropositive donors. CONCLUSION: Our results suggest that anti-RBD-S IgG reflect functional immune responses to SARS-CoV-2, but do not completely explain age- and sex-related disparities in COVID-19 fatalities.

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

A cautionary tale of false-negative nasopharyngeal COVID-19 testing.

There remains diagnostic uncertainty regarding the sensitivity of reverse transcription polymerase chain reaction in detection of SARS-CoV-2 from nasopharyngeal specimens. We present a case where two nasopharyngeal specimens were negative, followed by a positive sputum sample. Serial testing for COVID-19 is indicated in patients with high pretest probability of disease.