SARS-CoV-2 Continuous Genetic Divergence and Changes in Multiplex RT-PCR Detection Pattern on Positive Retesting Median 150 Days after Initial Infection

Abstract

Being in the epicenter of the COVID-19 pandemic, our lab tested 193,054 specimens for SARS-CoV-2 RNA by diagnostic multiplex reverse transcription polymerase chain reaction (mRT-PCR) starting in March 2020, of which 17,196 specimens resulted positive. To investigate the dynamics of virus molecular evolution and epidemiology, whole genome amplification (WGA) and Next Generation Sequencing (NGS) were performed on 9516 isolates. 7586 isolates with a high quality were further analyzed for the mutation frequency and spectrum. Lastly, we evaluated the utility of the mRT-PCR detection pattern among 26 reinfected patients with repeat positive testing three months after testing negative from the initial infection. Our results show a continuation of the genetic divergence in viral genomes. Furthermore, our results indicate that independent mutations in the primer and probe regions of the nucleocapsid gene amplicon and envelope gene amplicon accumulate over time. Some of these mutations correlate with the changes of detection pattern of viral targets of mRT-PCR. Our data highlight the significance of a continuous genetic divergence on a gene amplification-based assay, the value of the mRT-PCR detection pattern for complementing the clinical diagnosis of reinfection, and the potential for WGA and NGS to identify mutation hotspots throughout the entire viral genome to optimize the design of the PCR-based gene amplification assay.

Risk of reinfection and disease afterSARS‐CoV‐2 primary infection: Meta‐analysis

“…Of the 3282 papers initially retrieved, we included 91 cohort studies evaluating the onset of reinfection, severe and very severe/lethal COVID‐19 among 15,034,624 previously infected subjects (Figure 1), either among health care workers (27 publications; n = 37,598 2,8,9,15,17,24,36,38,42–59 ) or in the general population (67 publications; n = 14,997,026 3–7,10,12,16–23,25–27,43,49,60–106 ).…”

“…As shown in Table S1, most of the included studies ( n = 43) were carried out in Europe, 2,4–10,12,15,17,18,21,24,26,36,38,44,46–51,58,66,69,71,72,75,81,85–88,91–93,95,99,102 24 in the USA, 20,22,23,25,42,43,57,59,63,67,77,79,82–84,89,90,97,98,103,105–107 18 in Asia 3,16,19,27,45,52–54,56,60,62,64,65,70,73,74,100,101 and the remaining six in South America or South Africa 61,68,80,94,96,104 . The mean age of the participants ranged from 15 to 87 years, and the average follow‐up ranged from 29 to 371 days.…”

“…The mean age of the participants ranged from 15 to 87 years, and the average follow‐up ranged from 29 to 371 days. Overall, 38 publications strictly followed the CDC criteria to identify the reinfections and were classified at low risk of misclassification bias 10,16,19,20,22–24,26,36,44,45,48–50,54,56,58,59,68,71,72,75,84,86,89–91,94,99,100,102 . All but five studies ( n = 3,854,961) 66,71,88,91,96 were completed before the emergence of BA.1/B.1.1.529 (Omicron) variant.…”

“…In the 59 available datasets, 2,3,5,6,10,16–22,25–27,36,44,46–59,63–67,69,71–74,79,82,84–87,90,93–95,99–104 a total of 1380 severe COVID‐19 diseases were diagnosed among 5,006,604 subjects with a previous infection, corresponding to a pooled rate of 0.02% (95% CI: 0.00%–0.04%—Table 1). This rate did not substantially vary when only the studies with longer time lag were considered, while it increased—still, however, remaining below 0.1%—when only the studies that used CDC criteria to define reinfections were included (summary rate 0.07%; 0.04%–0.10%).…”

“…Overall, a total of 158,478 reinfections were recorded among the 15,034,624 subjects with a previous infection, 2–10,12,13,15–27,36,38,42–107 corresponding to a pooled rate of 0.97% (95% CI: 0.71%–1.27%—Table 1; Figure 2). The summary rate of reinfection rose to 1.07% (95% CI: 0.73%–1.46%) when only the 69 datasets with a more conservative time lag of ≥90 days between the two episodes were considered 3–6,8–10,12,18–23,25,26,36,42–44,46–50,52–56,59,61–65,67–69,72–84,86,87,89,91–96,98,101,103,105–107 and it slightly varied by reinfection definition criteria (0.93% vs 1.01% when considering studies with high vs low risk of misclassification). The risk of reinfection was higher among health care workers as compared to the general population (1.20% vs 0.90), and among females (0.79% vs 0.55% among males), although these estimates showed largely overlapping confidence intervals.…”