Afebrile patients with severe acute respiratory syndrome coronavirus 2 infection have a longer viral positivity duration: a retrospective analysis of 143 patients

Background A pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is on-going. Clinical characters of afebrile cases infected with SARS-CoV-2 remain poorly understood and informations are limited on the duration of SARS-CoV-2 viral positivity. Methods We performed a single-center retrospective study of 143 patients with SARS-CoV-2 infection in Beijing Ditan Hospital, Capital Medical University from January 26 to April 15


Background
The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic rapidly.On February 11, 2020, World Health Organization (WHO) o cially named the disease of the novel coronavirus disease 2019 (COVID-19) [1].By the end of the March, more than 7 million people worldwide have been diagnosed with the SARS-CoV-2 infection [2], which brought huge challenges and threats for both global health and economic development.Fever as the main clinical manifestation of SARS-CoV-2 infection is widely used in community and hospital emergency screening [3,4,5].However, we found that afebrile patients of SARS-CoV-2 infection count for a certain part in the process of diagnosis and treatment.What's more, the clinical characteristics and outcomes of these afebrile patients were rarely described until now.In this study, we did a retrospective study to compare the clinical characteristics and SARS-CoV-2 ribonucleic acid (RNA) positivity duration between afebrile and febrile patients with SARS-CoV-2 infection in Beijing Ditan Hospital.Besides, risk factors including clinical and laboratory data for the duration of SARS-CoV-2 RNA positivity were evaluated in 143 patients with SARS-CoV-2 infection.

Participants
From January 26, 2020 to April 15, 2020, a total of 237 patients were hospitalized and con rmed with SARS-CoV-2 infection in Beijing Ditan Hospital, Capital Medical University.We compared the characteristics between febrile and afebrile con rmed cases.For inclusion in the analysis, patients must be ≥18 years of age.Exclusion criteria: (1) Patients who were still hospitalized in Beijing Ditan Hospital, Capital Medical University until May 15, 2020.(2) The patients did not have complete clinical data, and in particular complete blood tests and peripheral blood T-lymphocyte subsets were not performed within 24 hours after admission.(3) Patients who had taken nonsteroidal anti-in ammatory drugs (NSAIDs) including aspirin, ibuprofen, acetaminophen and salicylate from herbal preps before admission.

De nition
The diagnosis and classi cation of COVID-19 was based on the latest Diagnosis and Treatment Guidelines for COVID-19 (Trial seventh version) [6].A con rmed case of COVID-19 was de ned as a positive result on high throughput seguencing or real-time reverse-transcriptase-polymerase-chainreaction (RT-PCR) assay of nasal and pharyngeal swab specimens [7].Mild type was de ned as having minimal symptoms and negative CT ndings.Common type was de ned as having fever, respiratory tract and other symptoms with pneumonia in imaging.Severe type was de ned as the presence of respiratory distress, respiratory rate ≥ 30 times/min; oxygen saturation (resting state) ≤ 93%; PaO2/FiO2 ≤ 300mmHg.Critical type was de ned as the presence of respiratory failure requiring mechanical ventilation, shock and other organ failure requiring ICU monitoring and treatment.In this study, we assessed the degree of severity of COVID-19 as mild (mild and ordinary type) vs. severe (severe and critical type) cases at the time of admission according to their condition mentioned above.Afebrile state was de ned as an axillary temperature less than 37.3 ℃ in the course of COVID-19.Leukopenia was de ned as a white blood count of less than 4000 cells per cubic millimeter.Lymphocytopenia was de ned as a lymphocyte count of less than 1000 cells per cubic millimeter.Thrombocytopenia was de ned as a platelet count of less than 100,000 per cubic millimeter.

Data collection
Specimens of the nasopharyngeal swab samples were collected every other day during hospitalization.SARS-CoV-2 RNA positivity duration time was calculated by an interval from the rst day with SARS-CoV-2 RNA tested positive to the rst day of SARS-CoV-2 returned to negative (at least consecutive two times of the RT-PCR negative results).The following parameters were collected from electronic medical records: (1) General conditions: gender, age, symptoms, time from illness onset to hospital admission and severity of illness; (2) Chest CT ndings within 24 hours after admission; (3) Laboratory characteristics: white blood cell, neutrophil, lymphocyte, monocyte, red blood cell, hemoglobin, platelet, brinogen, D-dimers, T lymphocyte, CD4 + T lymphocyte, CD8 + T lymphocyte, NK cell, B lymphocyte, albumin, lactic dehydrogenase, C-reactive protein, erythrocyte sedimentation rate, lactic acid, creatinine, sodium, potassium; (4) Clinical outcomes: duration of SARS-CoV-2 RNA positivity and length of hospital stay.The purpose of this study is to explore the characteristics of afebrile patients and observe the risk factors of viral RNA clearance within 30 days after illness onset.

Statistical analysis
The statistical analysis was performed utilizing SPSS software (Version 19.0).Continuous variables were expressed as median with interquartile range (IQR) and compared by Mann-Whitney U test.Categorical variables were expressed as number (%) and compared by Chisquare (χ²) test or Fisher's exact test between groups.Cox regression proportional-hazards model was applied to assess the hazard ratio (HR) of each variable for the duration of SARS-CoV-2 viral positivity, and signi cant risk factors whose HR was further adjusted with covariate analysis.All statistical tests were two-sided and a P < 0.05 was considered statistically signi cant.Kaplan-Meier method with a Log-rank test was performed to evaluate the signi cance of the duration of SARS-CoV-2 viral positivity between afebrile and febrile group.

Results
The selection process of included patients A total of 143 patients with con rmed SARS-CoV-2 infection were included in this study from January 26 to April 15, 2020 at Beijing Ditan Hospital, Capital Medical University, including 38 afebrile patients and 105 febrile patients.An overview of the selection process is presented in Figure 1.

Clinical characteristics of afebrile and febrile patients with SARS-CoV-2 infection at admission
There were no differences in age and gender between the two groups.Myalgia (P = 0.01) was more common in the febrile group than the afebrile group.There were no signi cant differences in respiratory symptoms including cough, sputum, shortness of breath, sore throat and rhinorrhea.11 patients (11/38, 29.0%) in the afebrile group showed normal chest CT on admission while 12 patients (12/105, 11.4%) in the febrile group showed normal chest CT on admission, the difference was statistically signi cant (P = 0.01).For overall patients, the median time from symptom appearance to hospital admission was 10 days (IQR, 6-13 days) and which had no signi cant difference between the two groups (P = 0.63).
Besides, no signi cant difference was observed in the proportion of severe cases at admission (P = 0.24) (Table 1).
The laboratory examination of the 143 patients is shown in Table 2. On admission, a total of 40 (28.0%)patients had leukopenia, 44 (30.8%) had lymphopenia and 8 (5.6%) had thrombocytopenia.78 patients (54.5%) with SARS-CoV-2 infection in this study had a dramatic loss of T lymphocytes, while 60 patients (42.0%) had decreased CD8+T lymphocytes and up to 105 patients (73.4%) had decreased CD4+T lymphocytes.Compared with febrile cases, afebrile patients had a signi cantly higher white blood cells (P = 0.02), total lymphocytes (P < 0.01), platelet count (P < 0.01), T lymphocytes (P < 0.01), CD4+ T cells (P < 0.01) and CD8+ T cells (P = 0.02).What's more, the plasma levels of C-reactive protein concentrations (P < 0.01) and sedimentation rate (P = 0.04) were lower signi cantly in afebrile patients than those in febrile patients.No signi cant differences were observed in NK cells (P = 0.93) and B lymphocytes (P = 0.67) between two groups.The median hospitalization time of 143 patients in our study was 21 days (IQR, 16-30 days).There was no statistical difference in length of hospital stay between two groups (P = 0.79).
To observe the occurrence of viral RNA clearance within 30 days after illness onset, univariate Cox regression proportional hazards model was used to evaluate the clinical and laboratory parameters on admission and the results showed a signi cantly shorter SARS-CoV-2 viral positivity duration was related to younger (HR = 0.99, P = 0.03), fever (HR = 0.54, P = 0.01) and higher count of platelet (HR = 4.39, P = 0.04).Multivariate Cox regression model showed fever (HR = 0.49, P < 0.01) and higher count of platelet (HR = 5.47, P = 0.02) were the predominant protective factors for a shorter SARS-CoV-2 RNA positivity duration (Table 4).Kaplan-Meier with Log-rank method was performed to evaluate the signi cance of fever to the SARS-CoV-2 viral positivity duration (Figure 2).

Discussion
The WHO had de ned COVID-19 as a pandemic on 11 March 202 [8], as a kind of coronavirus, the infectious number of SARS-CoV-2 were much higher than SARS-CoV in 2003 and MERS-CoV in 2012 [9,10].Temperature monitoring is currently widely used in community and hospital emergency settings as a major means of screening for SARS-CoV-2 infection, but a recent study has shown that the absence of fever in SARS-CoV-2 infection (42.8%) is much more frequent than that in SARS-CoV (1%) and MERS-CoV infection (2%) [9,10].Until now, reports on the clinical features of these non-febrile patients are rare.In this study, afebrile patients with SARS-CoV-2 infection accounted for 26.6% (38/143), which was lower than the previous studies [3,11].The different incidence rates may be explained by the different study populations, different de nitions of fever, different data and different prevalence among cities.
Fever is an evolutionarily conserved response of infection or injury and confers survival bene ts during damage.When the pathogen enters the body, the rst cells to detect foreign antigens are lymphocytes, which produce cytokines, such as interleukins-1 beta and tumor necrosis factor-alpha, to stimulate other immune cells and begin phagocytosis of invading organisms [12,13].In the early stages of phagocytosis, endogenous pyrogens are released, causing fever by raising the hypothalamic temperature-setting point in the anterior hypothalamus.Fever, as a consequence of infection and in ammation, also reduces the activity of pathogens, providing a more appropriate working environment for macrophages, lymphocytes and other immune cells, and it is now thought to be a protective response that can accelerate tissue recovery [14].Emerging evidence suggests that the increase in body temperature during fever is associated with the resolution of many infections and confers a survival advantage [15].In addition, fever enhances immune surveillance by promoting lymphocyte tra cking to lymphoid organs and in amed tissues during in ammation [15,16].That may explain the peripheral blood lymphocyte count in the febrile group was signi cantly lower than that in the afebrile group in our study.However, little is known about the regulation of lymphocyte by fever.In our study, the positive duration of SARS-CoV-2 RNA in febrile patients was signi cantly shorter than that in afebrile patients, and fever was markedly related to a shorter SARS-CoV-2 RNA positivity duration.Accordingly, we rst propose that fever may play a protective role during the process of SARS-CoV-2 infection.
Our study also found that patients in the febrile group had signi cantly lower platelet counts than those in the non-febrile group.However, patients with a higher count of platelet have a shorter duration of SARS-CoV-2 virial positivity.Beyond the main function in hemostasis and blood coagulation, platelets are currently thought to be an important component of the in ammatory and immune response in the processes of viral infections [17].It is susceptible to activation, damage, or degradation during severe infections or immune responses [18].Thrombocytopenia was documented in 44.8% of the SARS patients on presentation which was higher than our results in patients infected with SARS-CoV-2 [19].Suppression of bone marrow stromal cells, immune destruction, diffuse intravascular coagulation, platelet chemotaxis and peripheral migration, or phagocytosis by macrophages may be the potential mechanisms of the thrombocytopenia during viral infection [20].Increasing evidence supports the idea that platelets play a role in host defense against infections.Like traditional innate immune cells, platelets are mobilized adaptively from the bone marrow in response to infection and in ammation, being the earliest and most abundant cells preset at vascular sites of in ammation and release a broad-ranging of immune mediators microparticles and exosomes that modulate innate and adaptive immune cells [21].Evidences suggest that platelets could interact with viral pathogens directly [22,23].These functions are achieved through direct interaction with leukocytes, endothelial cells and via the release of soluble in ammatory mediators that enhance recruitment and activation of leukocytes [24,25].Besides, platelets also involve in phagocytosis by enhancing antigen presentation by antigen-presenting cells [26,27].The role of platelets in the clearance of virus was observed in respiratory syncytial virus infection by internalizing viral particles and by enhancing type I IFN production from peripheral blood mononuclear cells [28].Studies had shown that patients with thrombocytopenia in infectious diseases had a higher disease activity [29].We thus propose that low platelets induced immunode ciency in SARS-CoV-2 infection in part explain the negative predictive value of low or declining platelet count in our study.Increasing the understanding of immunoregulatory functions of platelets in viral infections will undoubtedly improve our knowledge on disease pathogenesis, clinical management, and therapeutic options.
In our study, leukopenia and lymphocytopenia occurred in 28.0% (40/143) and 30.8% (44/143) respectively.Lymphocytopenia was often detected in the infection of SARS-CoV [30], the exact mechanism is still being unclear.A study form Raymond SMW showed a signi cant decrease in white blood count and lymphocyte during the acute phase of SARS-CoV infection was found in 64% and 98% of patients respectively [31].Compared with SARS-CoV, patients infected with MERS-CoV have a relatively low probability of leukopenia (14%) and lymphopenia (34%) [32].Previous studies also showed that viral infections can lead to a down-regulation of lymphocyte subsets [33].More than 80% of patients have a decrease in CD4 + T and CD8 + T lymphocyte counts during the acute phase of SARS-CoV infection [34,35].In a recent study by Fan Wang et al., signi cant decreases in lymphocytes and their subsets were also observed in patients with SARS-Cov-2 infection [36], suggesting that SARS-CoV-2 infection may have a similar immunologic response to SARS infection.Although coronavirus is not known to productively infect T lymphocytes, altered antigen-presenting cell function and impaired dendritic cell migration resulting in reduced priming of T lymphocytes likely contribute to a fewer number of T lymphocytes [37].Despite extensive efforts, there is limited information available on the role of the antigen-speci c T cellmediated immune response to coronavirus including SARS-CoV-2.
Due to the sudden outbreak, the time of exposure to the patient's antigen and the onset of symptoms is relatively clear, we believe that this virus is the rst infection in these patients, and the immune response is also the primary immune response.This study provided data on the relationship between fever and viral clearance time of SARS-CoV-2 infection, and the result shows that the SARS-CoV-2 RNA positivity duration was signi cantly longer than that of the febrile patients.A higher count of platelet and fever are independent risk factors for the duration of SARS-CoV-2 nucleic acid positivity.Several limitations to this study must be acknowledged.First, it is a retrospective analysis of the data collected from a single center and, for this reason, our results may be biased.Second, the positive rate may be different between nasal swabs samples and sputum [38], so the bias might be introduced when comparing differences directly in viral RNA shedding between sputum versus nasal swabs.A large-scale cohort study and multi-center clinical data are still needed to further elucidate the features of SARS-CoV-2 infection and achieve a better understanding of the interactions between the virus and host response.

Conclusions
About a quarter of patients with SARS-CoV-2 infection did not experience fever in the course of SARS-CoV-2 infection.Compared with febrile cases, afebrile patients were more prone to develop asymptomatic and normal chest CT images.What's more, afebrile patients had a signi cantly higher white blood cell count, total lymphocytes, platelet count, T lymphocytes, CD8+ T lymphocytes and CD4+ T lymphocytes.
The SARS-CoV-2 virial positivity duration of the afebrile group was signi cantly longer than that in the febrile group.Fever and a higher count of platelet were the independent protective factors for a shorter SARS-CoV-2 RNA positivity duration.

Declarations Figures
Page 13/   The survival between groups with and without fever.Legends: Kaplan-Meier method with Log-rank test was performed to evaluate the signi cance of fever for the duration of SARS-CoV-2 viral positivity (P 0.01).

Figure 1 The
Figure 1