Epidemiological and clinical differences of coronavirus disease 2019 patients with distinct viral exposure history

ABSTRACT Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread from the early epicenter, Wuhan, to the rest of China, the virulence of SARS-CoV-2 might have evolved at different phases of the pandemic. We therefore compared the unique features among 62 coronavirus disease 2019 (COVID-19) inpatients who contracted SARS-CoV-2 in Wuhan (15 cases), exposed to the patients from Wuhan (26 cases), or acquired the disease without exposure to Wuhan patients (21 cases). Median incubation periods are 4.5 days (3–5) for Wuhan patients, 8 days (3–11) for those infected by Wuhan patients, and 12 days (7–13) for those without aforementioned experience. The disease onset dates are earliest for Wuhan patients and latest for those without exposure to Wuhan patients. Blood lymphocytes were lowest in Wuhan patients, lower in those affected by Wuhan patients, and modest reduced in remaining ones. Disease severity is worst for Wuhan patients, and modest for those without contact with Wuhan patients. Wuhan patients had longest (27 days, 18–28), those transmitted by Wuhan patients had intermediate (16 days, 8–23), and the rest of the patients had shortest (13 days, 8.5–22.5) hospital stay. Early viral exposure, older age, lymphocytopenia, and underlying conditions are risk factors which warrant aggressive intervention. Even though the virulence of SARS-CoV-2 appears decline over the course of serial transmissions, viral testing, contact tracing, social distancing, and face masking should be imposed on general public to contain viral dissemination from both symptomatic and asymptomatic patients with this highly contagious disease.


Introduction
An outbreak of respiratory illness caused by a novel RNA virus of Coronaviridae family was reported in Wuhan, Hubei, China [1][2][3]. Considering it a sister to severe acute respiratory syndrome coronaviruses (SARS-CoVs), International Committee on Taxonomy of Viruses assigned this novel virus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [4]. The World Health Organization (WHO) designated this disease as coronavirus disease 2019 (COVID-19) [5]. Because SARS-CoV-2 has plagued over 210 countries/territories/areas, WHO considered the public health threat posed by COVID-19 very high globally. As of 16 July 2020, WHO reported 13,378,853 confirmed cases and 580,045 deaths worldwide [6]. Due to exponential increase in cases and deaths, the US has become the country with the most confirmed cases and deaths of COVID-19. This widespread person-toperson transmission of SARS-CoV-2 has thus become a serious pandemic to threat public health globally. Navigating our responses in this uncharted arena and mitigating acquisition of viral infection and disease progression are global challenges.
China had 84,373 confirmed cases and 4,643 deaths with majority of them in Wuhan (50,333; 3,869) by 30 April 2020 [7]. To contain the rapid spread of SARS-CoV-2, Wuhan sealed its borders from the rest of China on 23 January 2020. The potential differences of epidemiological feature, clinical manifestation, response to medical intervention, and patient prognosis are unknown between patients with early infection inside Wuhan and subsequent transmissions beyond Wuhan. No direct comparison of earlier infected patients with later transmitted ones in the same medical setting(s) was reported. To elucidate the shared features and unique characteristics between these who infected with the virus in Wuhan before lockdown to those who contracted the virus later outside Wuhan, we retrospectively analyzed 62 COVID-19 inpatients treated from 20 January 2020 to 17 March 2020 at the Fifth Medical Center, Chinese PLA General Hospital, a designated medical center for care of COVID-19 patients in Beijing of China.

Study design and patients
This study was approved by the Medical Ethics Review Committee of the Fifth Medical Center, Chinese PLA General Hospital (2020018D). For this retrospective cohort study, we analyzed 62 Chinese patients with COVID-19 from 20 January to 17 March 2020 admitted at the Fifth Medical Center, Chinese PLA General Hospital. Pharyngeal swab specimens were obtained from all patients at admission and sent to Beijing Center for Disease Control for laboratory diagnosis. COVID-19 was diagnosed according to the WHO interim guidance and confirmed by at least two positive real-time reversetranscriptase polymerase-chain-reaction (RT-PCR) tests for viral nucleic acids [8][9][10].
We designated 24 January 2020 as illness onset cutoff date after lockdown of Wuhan implemented on 23 January 2020. We set patients' exposure history into three periods: (1) patients contracted the virus in Wuhan (Wuhan); (2) those exposed to Wuhan patients outside of Wuhan (Wuhan-related); and those acquired the disease outside of Wuhan without exposure to Wuhan patients (non-Wuhan). The clinical classifications are as follows [1]: Mild, The clinical symptoms are mild and no pneumonia manifestation can be found in imaging [2]. Ordinary, Patients have symptoms and pneumonia manifestation can be seen in imaging [3]. Severe, Meet any of the following: (a) Respiratory distress, RR≥30 breaths/min; (b) pulse oxygen saturation (SpO 2 )≤93% on room air at rest state; (c) arterial partial pressure of oxygen (PaO 2 )/oxygen concentration (FiO 2 )≤300 mmHg. Patients with >50% lesions progression within 24-48 hours in pulmonary imaging should be treated as severe cases [4]. Critical, Meeting any of the following: (a) respiratory failure occurs and mechanical ventilation is required; (b) shock occurs; (c) complicated with other organ failure that requires monitoring and treatment in intensive care unit. We cataloged mild and ordinary patients into nonsevere group and severe and critical patients into severe group on admission. Discharge criteria are as follows [1]: with normal body temperature for more than 3 days [2]; with significantly recovered respiratory symptoms [3]; lung imaging shows obvious absorption and recovery of acute exudative lesion [4]; virus clearance was evidenced by negative results of the nucleic acid tests of SARS-CoV-2 for consecutive two times (sampling interval at least 1 day) [11].

Data collection
Epidemiology, comorbidity, symptoms, vital signs, laboratory tests (complete blood count, blood chemical analysis, C-reactive protein [CRP], Procalcitonin [PCT], Interlukin-6 [IL-6], and Pro-brain natriuretic peptide [BNP]), chest computed tomographic (CT) scans, treatment measures such as antiviral, antibiotics, corticosteroid, respiratory support, and prognosis were all obtained from hospital electronic medical records. The incubation period was defined as the time from earliest possible exposure to the infected patients to the onset of illness. All the data were analyzed by a group of highly trained physicians and scientists.

Statistical analysis
Categorical variables were expressed as counts and percentages and analyzed using chi-square tests (sample size≥40 and expected frequency≥5) or Fisher's test (sample size<40 or Expected frequency <5), corrected α' = α/[k(k-1)/2] if comparing pairs within a group. Normality tests were conducted using Shapiro-Wilk test. Continuous variables were reported as the mean ± SD for normality and homogeneity of variances or median and interquartile range [IQR] for non-normality or non-homogeneity of variances. Continuous variables were compared using ANOVA for 3-group (SNK-q tests between-group comparisons) and t test for two-group comparison for normality and homogeneity of variances. With non-normality or nonhomogeneity of variances, Kruskal-Wallis was used for three-group (Wilcoxon rank sum test if comparing pairs within a group, corrected α' = α/[k(k-1)/2]) and Wilcoxon rank sum test for two-group comparison. Multiple linear regression examined how multiple independent variables were related to one dependent variable. Statistical analyses were performed with SAS software, version 9.4 (Cary, NC, USA), the statistical significance is defined as a two-sided α = 0.05.

Clinical differences between severe and nonsevere COVID-19 patients
Of the 62 patients, 43 (69%) were diagnosed as mild and 19 (31%) as severe on admission. Comparing with nonsevere patients, severe patients were older, and more likely had hypertension and diabetes (Table  5). Severe patients had more WBCs and neutrophils, higher D-dimer, CRP, IL-6, PCT and lactic acid, and fewer lymphocytes ( Table 6). The ratio of PaO 2 /FiO 2 was 205.5 (117-262) mmHg in severe patients and 350.7 (324-384) mmHg in nonsevere patients. More severe patients had expectoration and dyspnea. Chest CT scan showed obviously bilateral ground glass opacity and patchy shadows in severe patients ( Figure 3, Table 6). They developed septic shock, respiration failure, acute respiratory distress syndrome, acute cardiac injury, and had higher mortality rate. Antibiotics, glucocorticoid, gamma-globulin, noninvasive ventilation, and invasive mechanical ventilation were often used for severe patients (Table 6).  After treatment, most of the patients recovered and their inflammation indicators were gradually back to normal ranges.

Discussion
This retrospective cohort study documented the epidemiologic and clinical features of 62 COVID-19 patients treated in a medical center in Beijing of China. Compared with COVID-19 patients contracted the virus directly or indirectly from Wuhan-exported patients outside Wuhan, patients infected with the virus in Wuhan have shorter incubation period, severe clinical presentation, and worse prognosis. Severe patients are also associated with older age and comorbidities. COVID-19 is a respiratory viral disease presented with fever, dry cough, shortness of breath, and   pneumonia. Although most patients have mild symptoms, the disease can become very serious and even deadly for elderly people with preexisting health conditions [2,[12][13][14][15]. Globalization renders the world more vulnerable to the spread of infectious diseases. SARS-CoV-2 can be spread by asymptomatic, presymptomatic, and symptomatic carriers primarily via respiratory droplets during close face-to-face contact and therefore this virus spreads easily and sustainably in the community [16]. This ongoing pandemic was first documented in Wuhan City, Hubei Province, China. There are now much more confirmed cases and death outside of China than inside of China [6]. Many of published reports since the outbreak are about the patients contracted the virus inside Wuhan [2,12,17]. There are no comparison reports of the clinical manifestations and outcomes between early infected patients and later infected ones in same medical setting(s). We therefore investigated the potential distinct clinical features among the patients from Wuhan and outside Wuhan treated at our Hospital.
Our data indicate that patients contracted the virus outside Wuhan had longer incubation period, milder disease, better response to treatment, shorter hospital stay, and superior clinical outcome. As of 30 April 2020, Chinese Center for Disease Control and Prevention reported that Wuhan City's case death rate is 7.7% (3,869/50,333) compared with 2.3% (774/34,040) in the rest of the country [7]. Mizumoto and Chowell estimated that risk for death was up to 12% in Wuhan and 1% in the rest of China [14]. Even though flood of patients who overwhelmed the medical capacity certainly partially contributed to high death rate during the initial outbreak in Wuhan, all of the patients in this study were treated at the same hospital in Beijing. The duration of disease onset and hospital admission should reflect whether patients receive timely treatment. Since all of our patients   were admitted 4-5.5 days by average after disease onset, Wuhan patients' more serious condition was not attributed to delayed treatment. It is plausible that the patients exported from Wuhan might be less severe than those treated inside Wuhan. Nevertheless, two out of 15 (13.3%) Wuhan patients died while none of the 47 patients infected outside Wuhan died in this study. These data suggest that early generations of the viruses may be more potent than later descendants of the viruses. COVID-19 shares many similarities with Middle East Respiratory Syndrome (MERS) and SARS [18,19]. Like MERS-CoV and SARS-CoV, sequencing analysis revealed mutations and deletions on coding and non-coding regions of SARS-CoV-2 viruses and suggested genetic diversity and rapid evolution of SARS-CoV-2 viruses [18,20]. Genetic variants might influence the clinical presentation and spread of the disease [21] . It is plausible that potent variants are lethal to the hosts and therefore are less transmissible and eventually eliminated. On the other hand, less potent mutants cause mild disease and are easily transmitted person-to-person in the community. Therefore, potential genetic drift to less severe variant may be responsible for mild clinical outcome for patients infected with the virus outside of Wuhan in China. Viral samples should be sequenced to elucidate potential genetic alterations which may be responsible for their reduced virulence. SARS-CoV -2 virus can be transmitted quite efficiently by affected people who are just mildly ill or even asymptomatic or presymptomatic [12,[22][23][24]. Since MERS and SARS were spread only by symptomatic people with much less efficiency [25,26], COVID-19 is more contagious and much harder to contain [19]. Even though MERS and SARS have much higher case mortality rate, COVID-19 nevertheless has claimed much more lives overall. Corresponding counter measurements should be in place to combat this devastating pandemic.
Immune and inflammatory responses are prevalent in patients with initial viral and secondary bacteria and/ or fungal infections. Lymphocytopenia is more prominent in Wuhan-exported patients and severe patients. Adaptive immune system may be either suppressed or not activated during viral infection. The virus may destroy lymphocytes and/or lymphatic organs. T lymphocyte damage may contribute to disease deterioration [27]. The patients who contracted the virus in Wuhan or over 50 with hypertension and diabetes were more likely to become severe patients.
With no specific antiviral treatment for COVID-19, our management for mild patients was mainly supportive care. For severe patients, we repurposed existing antiviral medications and used prophylactic antibiotics to prevent complication. 96.8% patients were recovered and discharged by 17 March 2020. Prospectively, remdesivir and dexamethasone may hold promise for COVID-19 treatment [28][29][30][31][32][33]. Vaccination of general population is vital for prevention of recurrent COVID-19 pandemic.
Taken advantage of the same medical setting, we provide evidence that primary infected COVID-19 patients are more severe than secondary transmitted patients. We suggest that lymphocytopenia is a biomarker for poor prognosis. Older patients with comorbidities should be treated prophylactically for potential complications. Because our data were drawn from hospitalized patients, they would not necessarily be generalized to the public at large. This study of the limited number of patients only offers a glimpse at clinical manifestations of Covid-19, our preliminary findings should therefore be interpreted with caution. To facilitate preventing and mitigating this unprecedented pandemic, our findings are warranted validation from multicenter epidemiologic and clinical studies. Many patients with asymptomatic or minor manifestation were likely not documented. Due to their long latency, these undocumented patients may contribute to the rapid and extensive dissemination of SARS-CoV2 [23,24]. Extensive screening for virus carriers, contact tracing, face mask wearing, hand hygiene, and social distancing should be imposed on general public to contain viral transmission in community before the arrival of long-lasting measures, such as vaccination [34]. Underlying mechanism responsible for putative reduction of virulence associated with ensuing infections should be dissected.

Disclosure statement
No potential conflict of interest was reported by the authors.