In our study, we found distinction of severity of COVID-19 in different time period significantly. Compared with patients whose onset of illness began in January, the illness condition was improved in patients with onset of illness in February, which represented with lower mortality, lower rate of critical patients, lower level of neutrophil, D-miner, LDH, PCT, ALT, AST and a higher level of CD3 and CD4 cells count. Also, we observed significant increase in mild types of patients, asymptomatic patients and patients with normal CT scan whose illness onset began in February.
Increased neutrophil was positively related to poor outcome in coronavirus diseases. The study carried out by Wu et al demonstrated that neutrophilia is a risk factor related to the progression of ARDS and development from ARDS to death in COVID-19 patients [17]. The neutrophilia was also a risk factor for intubation in SARS patients [18]. The severity of lung damage positively correlated with neutrophils counts in the peripheral blood in patients with MERS [19, 20]. The neutrophil was able to produce chemokines and cytokines which can resist against the virus but at the same time it led to an excess inflammatory response that resulted in multiple organ injury. Whereas, D-miner was an independent risk factor for mortality in COVID-19 patients [17, 21]. Moreover, coagulation disorders was a displayed in the COVID-19 patients [22] and non-survivor had a higher level of D-miner, FDP and prolonged PT, APTT in COVID-19 patients [23]. Anticoagulant therapy might play the key role in relieving hypoxemia due to existence of micro-thrombosis in the lung of COVID-19 patients [24].The possible mechanism might be that the inflammatory cascade triggered by a virus and secondary infection led to injury of microvascular wall and release of procoagulant factors which resulted in ischemia and thrombosis. Evidence revealed that higher CD3 and CD4 T-cell counts protected patients from developing ARDS [17]. A recent study showed using electron microscopy that SARS-CoV-2 were in CD4 cells of COVID-19 patients, however, SARS-CoV-2 showed no productive replication [25]. This indicate that CD4 might control the replication of SARS-CoV-2. The incidence of secondary liver injury was positively related to poor outcome in COVID-19 patients [26]. A recent study showed that the liver injury was caused by activated cytotoxic T lymphocyte triggered by SARS-CoV-2 [27], other than drug-induced liver injury. Increased LDH was also associated with poor outcome in COVID-19 patients [17, 21]. Mild patients and asymptomatic patients increased with onset of illness in February, a recent study showed that COVID-19 patients who had mild or no symptoms, were dominant with virus in the upper respiratory tract [28, 29].
The mortality was reduced, symptoms and results of laboratory test were improved in patients with onset of illness in February compared with patients with onset of illness in January, however, there was no evident difference in age, sex, underlying diseases and environmental factors (district and seasonality) between them. Therefore, we speculate that the improved outcome might contribute to the diminishing virulence.
Recently, variants [30], mutations [31] and genetic diversity [32, 33] is found in SARS-CoV-2, which might be an explanations for diminishing virulence. A 382-nt deletion appeared during SARS-CoV-2 evolution [34], and a 29-nt deletion appeared during SARS-CoV evolution. Both of the deletions covered open reading frame 8 (ORF8). Evidence showed that the 29-nt deletion resulted in the decreased replicative ability of SARS-CoV in-vitro independent of the cell system [6]. The 382-nt deletion might also reduce virulence of SARS-CoV-2. An 81-nucleotide deletion had been found in ORF7 of SARS-CoV-2, leading to a 27 amino-acid in-frame deletion [35], which might also change pathogenicity of SARS-CoV-2. In the United States, researchers found discrepancy in mortality of COVID-19 patients between the East Coast and West Coast of America. They speculated that the discrepancy probably existed due to the change in amino-acid of viral spike protein in SARS-CoV-2 [36]. The study carried out by Li et al similarly found distinct pathogenicity of SARS-CoV-2 in vitro. The Vero-E6 cells were used to be infected with 11 SARS-CoV-2 viral isolates from COVID-19 patients. It showed huge discrepancy in pathogenicity on the Vero-E6 cells [37]. In addition, reduced virulence probably contributed to generation of antibody. A report showed that the improvement of clinical outcome in five COVID-19 patients who received an infusion of plasma from five patients who recovered from the COVID-19 [38].
In addition to diminishing virulence, some other factors which might have caused a difference in severity of COVID-19 in a different time period. At the early stage of the outbreak, the time from illness onset to hospital admission was longer, probably because of a shortage of medical resources and lack of awareness for COVID-19. It might aggravate illness after a few days of delay. Moreover, in the later time of outbreaks, more strict managements were employed [39], more number of Fangfang shelter hospital were established and the healthcare environment was improved. Furthermore, experiences and researches accumulated in early time played a crucial role in the improvement of clinical outcomes. (Fig. 3).
However, there were few limitations in our study. First, in the early stage of outbreak, many patients with clinical improvement were transferred to other isolated sites for further observation and waited for viral clearance, thus we did not obtain the time of viral clearance. Second, due to the retrospective study, some data in the analysis is missing. Third, some patients with onset of illness in February were not included as they were not discharged from the hospital yet. Moreover, our hospital was designated for to enroll the patients with severe and critical type patients with COVID-19, therefore the disease severity status and mortality in our research could not reflect the actual situation in Wuhan city. Nonetheless, our study was conducted at a single-center hospital with limited sample size.