key: cord-0857618-3o5c0l24
authors: Avadhanula, Vasanthi; Nicholson, Erin G; Ferlic-Stark, Laura; Piedra, Felipe-Andres; Blunck, Brittani N; Fragoso, Sonia; Bond, Nanette L; Santarcangelo, Patricia L; Ye, Xunyan; McBride, Trevor J; Aideyan, Letisha O; Patel, Kirtida D; Maurer, Lauren; Angelo, Laura S; Piedra, Pedro A
title: Viral load of SARS-CoV-2 in adults during the first and second wave of COVID-19 pandemic in Houston, TX: the potential of the super-spreader
date: 2021-02-15
journal: J Infect Dis
DOI: 10.1093/infdis/jiab097
sha: dedeae24b45344a8de55aac218f2ad5790d000ec
doc_id: 857618
cord_uid: 3o5c0l24

BACKGROUND: During the COVID-19 pandemic, a minority of index cases are associated with a majority of secondary cases suggesting that super-spreaders could drive the pandemic. We identified a phenotype in individuals with extremely high viral load who could act as super-spreaders. METHODS: Data were analyzed from individuals tested for SARS-CoV-2 from March 18 through August 15, 2020. Outcomes were compared using contingency table and quantile regression to test the equality of medians between the pandemic waves and by viral load groups. RESULTS: Of the 11,564 samples tested, 1,319 (11.4%) were positive for SARS-CoV-2. An increase in weekly median viral load occurred in the second wave of the SARS-CoV2 pandemic. This population was more likely to be women, outpatients, symptomatic and have an extremely high or high viral load. In patients with multiple RT-PCR positive tests, the duration of viral shedding was comparable between individuals with asymptomatic/mild and mild/moderate illness severity. CONCLUSIONS: We detected a small group of individuals with extremely high SARS-CoV-2 viral load with mild illness. We believe that these individuals’ characteristics could be consistent with the super-spreader phenomenon and that greater awareness of the social dynamics of these individuals is needed to understand the spread of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for of the coronavirus disease 2019 (COVID-19) [1] . Since the first documented case in Wuhan (Hubei province, China) in December 2019, SARS-CoV-2 has spread globally, leading to 58,570,555 cases and 1,386,596 deaths as of November 22 nd 2020 [2] . The SARS-CoV-2 pandemic has caused an unprecedented public health crisis similar to the 1918 influenza pandemic; albeit with important epidemiological differences such as a higher reproductive number (R 0 ) for SARS-CoV-2, and reduced hospitalization and mortality rates in children and young adults compared to older adults with COVID-19 [3] [4] [5] .

A number of factors likely contributed to this rapid global spread of SARS-CoV-2, including its high transmissibility and a high proportion of asymptomatic illness [6] . The virus itself seems well adapted to human spread due in part to high affinity binding of the SARS-CoV-2 virus to the angiotensin converting enzyme -2 (ACE-2) receptors on a variety of host tissues and organs promoting efficient intra-and interhost spread [7] [8] [9] . Environmental and other non-viral factors also play a role in transmission and disease.

Transmission by droplets or aerosols can occur more efficiently in enclosed settings or in poorly ventilated areas [10] [11] [12] . Environmental conditions such as air pollution and comorbidities conducive to more severe disease lead to incommensurate levels of infection and severe disease within members of resource-poor and marginalized communities [13] [14] [15] [16] Super-spreading events result in large outbreaks and sustained spread of disease from a few individuals [17, 18] . The duration of viral shedding and amount of virus a person sheds from the respiratory tract likely plays a vital role in transmission. Studies have shown that SARS-CoV-2 infected persons have peak viral loads 1-3 days before symptom onset, and can shed virus for three or more weeks [19, 20] .

Similarly, both symptomatic and asymptomatic individuals can transmit the virus efficiently and can have prolonged viral shedding [21] [22] [23] . Consequently, it is important to understand the role of viral load and A c c e p t e d M a n u s c r i p t 4 viral shedding in the transmission of SARS-CoV-2. In this report, we present data related to viral load, viral shedding and illness during two separate waves of the SARS-CoV-2 pandemic in Houston, Texas, USA. We describe a group of individuals with an extremely high viral load who have the potential to be super-spreaders within the community and become major drivers of the pandemic. Relative copy number for N1 and N2 were extrapolated later from a standard curve run on separate plate using six 10-fold serial dilutions (1x10 6 to 1x10 1 copies/reaction) of a plasmid containing a complete N gene (IDT technologies).

Comparison categories. We defined the SARS-CoV-2 pandemic into two waves. The first wave occurred from March 18 to May 31, 2020, and the second wave from June 1 through August 15, 2020. A priori, we considered individuals as potential super-spreaders if they had either extremely high or high viral load, and/or prolonged viral shedding. Extremely high or high viral load was defined as Ct values <16 or 16 to <21, respectively. Prolonged viral shedding was defined as having two or more positive RT-PCR tests from an individual on two or more different days. The three categorical comparisons were 1) the first versus second wave, 2) viral load groups classified by their Ct value as extremely high (<16), high (16 to <21), medium (21 to <31) and low (31 to <40), and 3) individuals with single versus two or more RT-PCR positive tests. For all the data analysis involving viral load and viral shedding, N1 Ct value was used.

Statistical analysis and generation of the graphs were carried out using Stata 16.0 (Stata Corp, College Station, Texas). Continuous variables were summarized as median with interquartile range (IQR), or geometric mean with confidence interval (CI) and categorical variables as frequency with percentage of total. Demographic characteristics and RT-PCR outcomes were compared between waves, viral load groups, and single vs. multiple samples with the use of contingency M a n u s c r i p t 6 Two summary statistics: median duration and cumulative percent at given duration were derived from the serial Ct values to describe viral load kinetics by group and analyzed using quantile regression. No multiple-comparisons adjustments were made to account for multiplicity in testing. Two-sided P values were reported, with P < 0.05 considered significant. varying viral loads. The extremely high (N1 Ct <16.0 or >9.74 log 10 copies/mL), high (N1 Ct 16 to <21 or 7.97 to 9.73 log 10 copies/mL), medium (N1 Ct: 21 to <31 or 5.70 to 7.96 log 10 copies/mL) and low (N1 Ct: 31 to <40 or 2.38 to 5.69 log 10 copies/mL) viral load. Interestingly, during the first wave the weekly viral load (median Ct was 21.3) was highest at CDC week 12, which was followed by the highest positivity A c c e p t e d M a n u s c r i p t 7 rate of 15% at CDC week 15. Similarly, during the second wave the weekly viral load was highest (median Ct was 21.7) during CDC week 25 and was followed by the highest positivity rate of 20% at CDC week 27.

Phenotype of individuals in the second SARS-CoV-2 pandemic wave. Individuals during the second pandemic wave (n=751) were significantly more likely to be female, been seen at a clinic, have lower median Ct values, and be in the extremely high or high viral load groups ( Table 1) . Although not statistically significant, there was a trend for higher proportion of Hispanics and individuals with nocomorbidity to be positive during the second wave. The higher median viral load observed in the second wave was in the asymptomatic to mild and mild to moderate disease severity categories (Figure 3) . The mean viral load in the second wave was ~ 7 Ct greater, i.e. there was 128-fold higher viral load than the first wave (n=77). The phenotype identified during the second wave suggested that healthy females, many asymptomatic, were contributing to the high viral load being shed in the community.

To further define the phenotype of SARS-CoV-2 infected individuals, we analyzed the population by the four viral load categories based on the Ct distribution of the N1 primer ( Table 2) . Individuals in the extremely high and high viral load groups had on average 20 and 16 Ct difference, respectively, when compared to the low viral load group.

This represents an average of approximately 1 x 10 6 and 6.6 x 10 4 fold-higher viral load in the extremely high and high viral load groups compared to the low viral load group. Significant differences were seen for illness severity, site of testing, number of RT-PCR tests performed. A non-significant trend was observed for race and ethnicity ( Table 2 ). The extremely high viral load group phenotype was, in part, characterized by individuals presenting to the clinic or hospital with symptomatic illness, while individuals in the high viral load group were more likely to be asymptomatic at the time of diagnosis, evaluated at the occupational health clinic, and be RT-PCR positive two or more times.

A c c e p t e d M a n u s c r i p t 8 Phenotype of individuals with two or more positive SARS-CoV-2 PCR tests. A priori, we considered individuals with two or more positive RT-PCR tests as those likely to shed the virus for longer duration compared to individuals with a single positive RT-PCR test ( Table 3) . The individual phenotype with two or more positive RT-PCR tests were more likely to be asymptomatic or have a mild illness at the time of the initial diagnosis, be seen at the occupational health clinic and have lower median Ct values, which translates to a 7 to 8-fold higher viral load compared to individuals with a single positive RT-PCR test. The median duration of viral shedding for the extremely high (n=10), high (n=42), medium (n=28) and low (n=38) viral load groups was 28.5, 25, 21.5, and 26.5 days, respectively. For each viral load category, the duration of viral shedding was comparable between individuals with asymptomatic to mild and mild A c c e p t e d M a n u s c r i p t 9 to moderate illness severity. Remarkably, the low viral load group had limited fluctuation in their viral load even though they experienced prolonged viral shedding.

The present study describes the first two waves of the SARS-CoV-2 pandemic in Houston, TX, USA. We observed an increase in the weekly median viral load that predated the onset of each wave by approximately two weeks. This was more evident during the second wave when the city of Houston was reopening from the initial lockdown. As the weekly median viral load increased, the percent positivity also increased with peak activity offset by two weeks. Similarly, as the weekly median viral load levels decreased, the percent positivity also decreased. This fluctuation in the weekly median viral load was, in part, the result of a subset of individuals detected with extremely high and high viral load levels. Individuals with extremely high and high viral load represented 7.1% and 20.8%, respectively, of the RT-PCR positives in our surveillance study. Such high viral load levels are infrequently observed with other respiratory viruses, even in children [25, 26] . Our data support the concept that these individuals are potential super-spreaders for SARS-CoV-2 and major drivers of the pandemic waves. Recent studies document that a minority of index cases are associated with a majority of the secondary cases, consistent with the concept of the super-spreader being a major catalyst of the SARS-CoV-2 pandemic [27] .

The extremely high viral load group's phenotype was characterized by individuals presenting to the clinic or hospital with a mild symptomatic illness. These observations were similar to other studies where there was no relationship of high viral load to severity of disease [20] [21] . The median duration of viral shedding varied between 25-28.5 days for extremely high and high viral load groups when limited to individuals with 3 or more RT-PCR positive samples, and was greater than the median duration of 14.5 days observed in a systematic review [28] . In our individuals with prolonged viral shedding, the period with the highest viral load occurred early in their illness when they are most likely to be infectious. Our observations are consistent with reports that describe viral load shedding kinetics with the highest levels A c c e p t e d M a n u s c r i p t 10 occurring several days prior to and 7 to 10 days after illness onset [19, 29] . In addition, the ability to isolate infectious virus occurred only in individuals with very high viral levels and within the first 10 days after illness onset [30, 31] .

In our study, a majority of individuals were evaluated at outpatient clinics and tested two or more times for SARS-CoV-2. This group appeared to maintain medium to high viral load for about 10 days from their first RT-PCR positive test suggesting they had the potential to remain infectious during this time period. In a recent report that described the transmission dynamics of SARS-CoV-2 in two Indian states, approximately 8% and 20% of the index cases were responsible for transmitting approximately 60% and 40%, respectively of the secondary cases [32] . Impressively, no positive cases were detected from contact tracing of approximately 70% of the index cases. Comparable results were reported from Shenzhen, China where 9% of index cases were responsible for 80% of secondary infections [33] . It is tempting to speculate that our population of extremely high (7.1%) and high viral load (20.1%) could be responsible for the majority of secondary cases.

The overall phenotype seen in the second pandemic wave shifted to women with no reported comorbidity who were overrepresented in the extremely high or high viral load groups. It is apparent that extremely high and high viral loads do not translate to disease severity. Many were asymptomatic or had mild illness indicating that without appropriate viral detection, social distancing and quarantine, individuals who have extremely high or high viral load will be able to spread SARS-CoV-2 and sustain the current COVID-19 pandemic.

In addition to viral load, it is essential to evaluate other mechanisms potentially contributing to efficient This study has some limitations. First, the population reported was not representative of the community but rather of individuals who worked within the medical center or used the healthcare services of the medical center. Although these individuals reside within the greater Houston area, there may be confounders that place them at greater risk for SARS-CoV-2 infection. Secondly, the population represents a cross-sectional observational cohort rather than a prospective cohort, which limits the available clinical data. However, in approximately half of the positive individuals we were able to obtain two or more time points with metadata, which add to the strength of our clinical findings. Lastly, the viral kinetic data were limited to individuals who came in primarily to determine when they were negative for SARS-CoV-2. Although the timing of the second and subsequent RT-PCR tests were not performed within a set protocol, they were generally performed every 7 to 14 days until they cleared their infection.

In summary, we detected a marked increase in the median viral load of SARS-CoV-2 infected individuals during the second wave of the pandemic. The extremely high and high viral load groups in general were asymptomatic or had mild clinical illness. The duration of the high viral load and the mild nature of the illness suggest many individuals go undiagnosed. Greater awareness of the social dynamics of these individuals is needed to understand their potential to be super-spreaders of SARS-CoV-2.

M a n u s c r i p t M a n u s c r i p t M a n u s c r i p t 22 

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