Viral Dynamic Surveillance in COVID-19 Patients: A Cohort Study

Background Coronavirus disease 2019 (COVID-19) is a potentially fatal pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially those of novel SARS-CoV-2 variants and infection has affected over 700 million people globally. Methods This retrospective, descriptive study included 118 patients admitted with SARS-CoV-2 infection as confirmed by real-time reverse transcription polymerase chain reaction. Results The median duration of detectable SARS-CoV-2 infection in patients with high ALT, AST, and PLT/LYMPH, or low CD4+, CD8+, and PLT/MONO was considerably longer. In the risk factor model, multivariate analysis was performed for the estimation of ALT (HR, 0.54; 95% CI, 0.36-0.81), AST (HR, 0.56; 95% CI, 0.34-0.93), CD4+ (HR,0.77; 95% CI, 0.48-1.24), CD8+ (HR,0.64; 95% CI, 0.37-1.11), PLT/LYMPH (HR, 1.16; 95% CI, 0.76-1.77), and PLT/MONO (HR, 0.64; 95% CI, 0.43-0.94). Conclusions The longer viral RNA duration was associated with a higher International Prognostic Index score (p = 0.0013), demonstrating for the first time that multivariate features of the bioindicators closely associated with SARS-CoV-2-infected patients clear the virus.


Introduction
First detected in December 2019, a highly contagious pneumonia, called novel coronavirus disease 2019 (COVID- 19), has spread worldwide and caused a global public health crisis [1,2]. The World Health Organization (WHO) reported more than 248 million confirmed cases of COVID-19, which have resulted in more than five million fatalities globally as of November 3, 2021 [3]. The causative agent was identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus, in bronchoalveolar lavage fluid samples from patients [1]. SARS-CoV-2 is distinct from severe acute respiratory syndrome coronavirus (SARS-CoV), which arose in Guangdong, China, in 2002 [4], and led to more than 8,000 infections and 700 deaths globally from 2002 to 2003 [5], and Middle East respiratory coronavirus (MERS-CoV), which was first reported in Saudi Arabia in 2012 [6], causing infection-related mortality rates of 41% and 35% in Saudi Arabia and globally, respectively [7]. SARS-CoV-2 belongs to the orthocoronavirinae subfamily; its highest genetic similarity is to a bat coronavirus, Bat CoV RaTG13, with which it is 96% identical at the wholegenome level [8,9]. Further investigation is needed for definitive determination of the origin of SARS-CoV-2.
COVID-19, which has a clinical profile like severe acute respiratory syndrome (SARS), can result in death from underlying disease or acute respiratory distress syndrome due to lung injury [1-2, 4-5, 8]. Early evidence linked the spread to seafood markets, but human-to-human transmission has now been confirmed [2,10]. Characterization of    BioMed Research International the high-affinity interaction between SARS-CoV-2 and angiotensin-converting enzyme-2 (ACE2), a cellular receptor for SARS-CoV and SARS-CoV-2, revealed that it enables efficient transmission by direct contact or via respiratory droplets [11,12]. The presence of inframicrobes in faeces and blood has been confirmed via polymerase chain reaction (PCR) [13], indicating the danger of aerosol generation and propagation of COVID-19 in small, enclosed spaces.
Notably, different SARS-CoV-2 variants defining nonsynonymous mutations have successively emerged worldwide [14][15][16][17][18]. SARS-CoV-2 is one of the deadliest viruses that infect both humans and animals including cats [19,20], dogs [20,21], and mink [22], then infected animals can transmit the virus back to humans [21,22]. Although the mechanism of animal-to human transmission is unclear, evidence suggests that these variants exhibit superior cross-species transmission capacity. Compared with the wild-type strain Wuhan-Hu-1, recently identified variants B.  [17,25], and B.1.617 in India [18] exhibit robust transmissibility owing to residue changed of these variants. These mutations, contributing to stabilize the complex structure of spike protein with ACE2 [26] and be responsible for SARS-CoV-2 immune escape [27], was the worldwide spread of a pandemic.

Procedures.
Throat swabs, sputum, faeces, urine, semen, vaginal discharge, and milk specimens were collected from patients. RNA was extracted from the samples using the Nucleic Acid Isolation Kit (Magnetic Beads) (SDK60105; Jiangsu, China), according to the manufacturer's protocol. The RNA was retained after removing human nucleic acids. We detected SARS-CoV-2 using primers and probes for two envelope genes of interest, open reading frame 1ab (Orf1ab), and nucleocapsid protein (N). The nucleic acid amplification reaction buffer, enzyme mixture, Orf1ab/N reaction solution, and RNA suspension were placed into a centrifuge tube. In addition, positive and negative controls were included to verify diagnostic reliability. The real-time reverse transcription PCR (RT-PCR) assay was performed using a SARS-CoV-2 nucleic acid detection kit according to the instructions provided by Guangzhou Daan Biotechnology Co, Ltd. The RT-PCR amplification protocol comprised reverse transcription at 50°C for 30 minutes and 95°C for 5 minutes, followed by 45 cycles of denaturation at 95°C for 10 seconds, and annealing, extending, and fluorescent signal detection at 55°C for 40 seconds.
The RT-PCR assay was considered negative if the cycle threshold value (Ct-value) was >38 and positive if the Ctvalue was ≤35. Samples were retested if the Ct-value was >35 and ≤38. The results were considered positive if the Ct-value remained between 35 and 38 after retesting.
In conclusion, we proposed a multivariable Cox proportion hazard regression model, where the prognostic index resulted from a combination of factors. Collectively, these parameters suggest the duration of viral shedding and disease progression.

Discussion
In the present study, we systematically compared the dynamics of SARS-CoV-2 infection and disease progression via 118 patients to characterize the factors that influence host defense against SARS-CoV-2.
Previous studies showed that cellular immune response, in addition to the humoral immune response, plays an important role in T-cell immunity-mediated virus clearance [28][29][30]. We further investigated the kinetic determinants of virus shedding and found that higher frequencies of CD4+ and CD8+ T-cells may be associated with better virus clearance. Clinical strategies to improve the outcomes of patients with COVID-19 who receive antiviral therapy should consider their CD8+ T-cell counts. Our findings suggest that CD4+ and CD8+ T-cells play an important role in restricting virus replication and suppressing the progression of COVID-19 and can be used to assess prognosis. Lower CD4+ and CD8+ T-cell levels were associated with an increased risk of a worse clearance of the virus.
Platelets showed important and distinct characteristics in bacterial [31], malaria [32], and virus [33] infection. Large scale clinical studies in many countries and centers suggested that thrombocytopenia was significantly associated with poor prognosis, but the mechanism was unknown [34]. We found no difference in the duration of viral BioMed Research International shedding in those platelets less than 100 × 10 9 /L vs. those platelets 100 × 10 9 /L and more, however, we also conducted prespecified secondary analysis to elucidate the platelets were associated with risks potentially related to viral clearance. Higher PLT/LYMPH and lower PLT/MONO ratio were associated with an increased risk of elongation time for viral shedding. Six of the risk factors seemed to have much impact on the duration of viral RNA shedding, however, PLT/LYMPH and PLT/MONO ratio were two significantly high-risk factors, which may be attributed to the fact that platelets owned various immunological functions and exerted its regulatory role on the lymphocytes and monocytic cells.
Most patients with COVID-19 suffer from liver dysfunction [35], and we observed that low levels of alanine aminotransferase and aspartate aminotransferase correlate with a short duration of SARS-CoV-2 infection. Therefore, alanine aminotransferase and aspartate aminotransferase levels may be a prognostic marker in patients with COVID-19.
This study was the first to explore the potential prognostic value of these risk factors assessed during COVID-19 pandemic, SARS-CoV-2-infected patients. These risk factors considered together suggested that the prognosis for patients with COVID-19 should be regarded as an accurate prediction by the proportional hazards regression model. Consequently, it is significantly important to group by prognostic index for monitoring the changes in viral dynamics and guiding clinical therapies.

Patents
This was a retrospective, descriptive study, and no patients were directly involved in the study design, setting, research questions, or outcomes measures. No patients were asked to advise on interpretation or writing up of results. Demographics and baseline characteristics of the 118 patients with COVID-19 were summarized in Table 1.

Data Availability
Epidemiological, clinical, and laboratory characteristics and treatment and outcome data were obtained through data collection forms from hospital electronic medical records.

Ethical Approval
The study was conducted according to the guidelines of the

Conflicts of Interest
The authors declare no conflicts of interest.