From March 1, 2020 through May 15, 2020, 19,431 specimens were tested by real time PCR at the Nevada State Public Health Laboratory of which 1264 specimens were deemed positive. By analysis of N1 gene detection by real time PCR data, the average Ct value and standard deviation of such specimens was found to be 27.55±6.11. Of these positive cases, 23 were followed and were found to be associated with cases with no symptoms. The mean Ct value and standard deviation of these 23 cases was found to be 29.63±3.81. Of the 1264 positive specimens, 30 were from cases that involved COVID-19 related fatality of the tested patient. The average Ct value and standard deviation of these 30 fatal cases was 23.36±5.73. The difference between mean Ct values of fatal cases and all cases (4.19) is considered statistically significant (p=0.0004, two-tailed test) as well as the differences between the means of fatal cases and asymptomatic cases (6.27) (p=0.0004). However the difference in mean Ct values between asymptomatic cases and all cases (2.08) may not be (p=0.103) (Summary of mean values in Table 1).
N1 target human RP n Asymptomatic 29.63 26.35 23 All cases 27.55 26.11 1264 Fatal 23.36 24.79 30 N1 target: analyte-specific (SARS_CoV-2) target of EUA CDC RT-PCR; Human RP: human RNASeP target, internal control of EUA CDC RT-PCR
Table 1. Mean Ct values of cases
For each collected specimen, the real-time PCR test to assess for the presence/absence of analyte (SARS-CoV-2) RNA includes a co-analysis for detection of human RNAse P RNA (RP) by real-time PCR. Mean RP Ct values and standard deviations of all cases, asymptomatic cases, and fatal cases respectively were 26.11±2.29, 26.35±2.29 and 24.79±2.62. The differences of 1.32 between the mean Ct values of fatal cases and of all cases is deemed statistically significant according to two-tailed test (p=0.006). Observing this, we sought to determine whether decreasing RP Ct values demonstrated a correlation to decreased analyte (N1) Ct values for specimens overall. We calculated the coefficient of determination (R2) for N1 Ct values vs. RP Ct values and found it to be 0.0187 (n=300 consecutively tested specimens), indicating a very weak relationship between the two values overall.
With regard to the differences in the amount of viral genomic material associated with swabs based on disease outcome, we sought to determine whether there are any genetic differences in the viruses associated with fatalities vs. those generally detected. To accomplish this, we performed sequencing on the virions associated with 16 of 33 cases which involved fatality (selected at random), and to compare those sequences to sequences generated from 154 other cases selected randomly from positive cases from multiple locations throughout Nevada. The sequences of virus were assessed for the presence of polymorphisms that correlated with disease severity or Ct value.
Virus associated with fatality or low Ct showed a variety of genotype, with no singular strain / sequence associated with all such cases. Only two mutations were found that were exclusively associated with fatal cases. As shown in Figure 1, one branch of epidemiologically linked cases showed only fatal cases (011, 015, 016, and 023). The four cases were deaths that occurred within a 2-week period among residents of the same senior living community. These cases were associated with low Ct value in three of four instances (17.60, 18.35, 16.16 and 29.47). All four cases had two base changes relative to the reference sequence that were not seen in any of 156 other cases sequenced (97 shown). The first change was at base 18377 (C>T) which results in a change of an alanine to a threonine at amino acid position 6038 of the orf1ab polyprotein gene. This location denotes the reading frame of a 3’ to 5’ RNA exonuclease enzyme. The second change seen among these cases is at position 28187 (T>C) which results in a change of leucine to serine at amino acid position 95 in the orf8 gene. The alteration at base 18377 was seen in four other sequences submitted to NEXTSTRAIN.org out of 3104 total genomic sequences submitted as of June 20, 2020. The T>C base change seen at 28187 was not previously observed according to NEXTSTRAIN.org. Each of the four cases harbored virus that had the D614G alteration as well, which has been associated with higher infectivity and poorer outcomes[3, 4, 5, 6]. All four cases involved people over the age of 77 at the same long-term living facility.
Characteristics of Viral Specimens Collected from Asymptomatic and Fatal Cases of COVID-19
Abstract: We sought to determine the characteristics of viral specimens associated with fatal cases, asymptomatic cases and non-fatal symptomatic cases of COVID-19. This included the analysis of 1264 specimens found reactive for at least two SARS-CoV-2 specific loci from people screened for infection in Northern Nevada in March-May of 2020. Of these, 30 were specimens from fatal cases, while 23 were from positive, asymptomatic cases. We assessed the relative amounts of SARS-CoV-2 RNA from sample swabs by real-time PCR and use of the threshold crossing value (Ct). Moreover, we compared the amount of human RNAse P found on the same swabs. A considerably higher viral load was found to be associated with swabs from cases involving fatality and the difference was found to be strongly significant statistically. Noting this difference, we sought to assess whether any genetic correlation could be found in association with virus from fatal cases using whole genome sequencing. While no common genetic elements were discerned, one branch of epidemiologically linked fatal cases did have two point mutations, which no other of 156 sequenced cases had from northern Nevada. The mutations caused amino acid changes in the 3’-5’ Exonuclease protein (ExoN), and the product of the gene, orf8.
|Citation:||Andrew J. Gorzalski, Paul Hartley, Chris Laverdure, Heather Kerwin, Richard Tillett, Subhash Verma, Cyprian Rossetto, Sergey Morzunov, Stephanie Van Hooser, Mark W. Pandori. Characteristics of Viral Specimens Collected from Asymptomatic and Fatal Cases of COVID-19[J]. The Journal of Biomedical Research. doi: 10.7555/JBR.34.20200110|