key: cord-347058-kejcwlng
authors: Akbari, Hamed; Tabrizi, Reza; Lankarani, Kamran B.; Aria, Hamid; Vakili, Sina; Asadian, Fatemeh; Noroozi, Saam; Keshavarz, Pedram; Faramarz, Sanaz
title: The role of cytokine profile and lymphocyte subsets in the severity of coronavirus disease 2019 (COVID-19): A systematic review and meta-analysis
date: 2020-07-29
journal: Life Sci
DOI: 10.1016/j.lfs.2020.118167
sha: 
doc_id: 347058
cord_uid: kejcwlng

AIMS: This study aimed to make a comparison between the clinical laboratory-related factors, complete blood count (CBC) indices, cytokines, and lymphocyte subsets in order to distinguish severe coronavirus disease 2019 (COVID-19) cases from the non-severe ones. MATERIALS AND METHODS: Relevant studies were searched in PubMed, Embase, Scopus, and Web of Science databases until March 31, 2020. Cochrane's Q test and the I(2) statistic were used to determine heterogeneity. We used the random-effect models to pool the weighted mean differences (WMDs) and 95% confidence intervals (CIs). KEY FINDINGS: Out of a total of 8557 initial records, 44 articles (50 studies) with 7865 patients (ranging from 13 to 1582), were included. Our meta-analyses with random-effect models showed a significant decrease in lymphocytes, monocyte, CD4+ T cells, CD8+ T cells, CD3 cells, CD19 cells, and natural killer (NK) cells and an increase in the white blood cell (WBC), neutrophils, neutrophil to lymphocyte ratio (NLR), C-reactive protein (CRP)/hs-CRP, erythrocyte sedimentation rate (ESR), ferritin, procalcitonin (PCT), and serum amyloid A (SAA), interleukin-2 (IL-2), IL-2R, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (INF-γ) in the severe group compared to the non-severe group. However, no significant differences were found in IL-1β, IL-17, and CD4/CD8 T cell ratio between the two groups. SIGNIFICANCE: Decrease in total lymphocytes and lymphocyte subsets as well as the elevation of CRP, ESR, SAA, PCT, ferritin, and cytokines, but not IL-1β and IL-17, were closely associated with COVID-19 severity, implying reliable indicators of severe COVID-19.

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f pediatric or pregnant cases due to the diverse presentation of COVID-19 in these groups, (2) inadequate information on inflammatory-related laboratory parameters in either severe or non-severe disease groups, (3) coronavirus strains other than COVID-19, (4) and studies with unusable data. Nonetheless, the diagnostic criteria for COVID-19 were explained on the basis of laboratory approved SARS-CoV-2 infection. If two or more studies were published by the same authors or institutions, only the study having the largest sample size was selected.

The data from the incorporated studies were extracted by two reviewers (SV and SF)

independently. Also, a third reviewer (RT) was used to solve any arisen argument. The details of each study were collected which involve author, publication date, study location, study design, sample size, sample characteristics (age, gender, comorbidities), exposure characteristics (study definition of severity of COVID-19, the timing of classification of disease severity [on admission or otherwise], number of cases with non-severe COVID-19, number of cases with severe or critical COVID- 19) , the timing of blood sample collection (on admission or otherwise).

Moreover, inflammatory-related laboratory factors, cytokines, lymphocyte subsets, and CBC indices were grouped by COVID-19 severity (mean [SD] ) and finally, all the extracted data were transferred into Microsoft excel. Furthermore, through re-checking the primary studies, as well as discussions, any inconsistencies in the extracted data were resolved. It is worth mentioning that, using web plot digitizer online software, some graph data were converted to numerical data (https://apps.automeris.io/wpd/). In case the relevant data were missing, authors of selected studies were contacted via email. Also, it should be noted that due to inaccuracies in the research methodology for some of the studies, we reported the type of study in some articles, especially those submitted in the medRxiv, by inference.

The included studies differed in the way they defined patients' disease status, and classified the disease into 'mild, moderate, severe and critical', 'ordinary and severe/critical', 'common and severe', and 'non-severe and severe', categories. The first outcome measure adopted was severe (including both severe and critical cases) vs. non-severe disease. The definition provided J o u r n a l P r e -p r o o f patients as shown by their symptoms and the imaging examination; (3) severe: patients with any of the following factors: (i) respiratory rate equal to or higher than 30/min; (ii) resting pulse oxygen saturation (SpO 2 ) equal to or lower than 93%; (iii) oxygen partial pressure (PaO 2 ) / fraction of inspired oxygen (FiO2) equal to or lower than 300mmHg (1mmHg = 0.133kPa); (iiii) imaging process showing a 50% progression in multiple pulmonary lobes of a lesion in hours; (4) critical: patients with any of the following factors: (i) the need for mechanical ventilation in case of respiratory failure (ii) shock; (iii) admission to the intensive care unit (ICU) due to simultaneous failure in another organ. It is noteworthy that mild or moderate patients were included in the non-severe group, while severe or critical patients were included in the severe one. The Newcastle-Ottawa Scale was used to evaluate quality, and moreover, assessment scores of 0-3, 4-6, and 7-9 represented poor, fair, and good studies, respectively.

Additionally, discrepancies were resolved through consensus.

All statistical analyses were conducted using STATA version 12.0 (Stata Corp., College Station, TX). Laboratory factors were considered as the mean (SD) difference with 95% confidence intervals (CIs) between the severe group and the non-severe group. To pool the mean differences (SD), weighted mean difference (WMD) statistic with the random-effect model (DerSimonian-Laird method) were used. Cochrane's Q test or the I 2 statistic was used to assess heterogeneity among included studies. I 2 above 70% and Cochrane's Q test with P < 0.05 was considered as the existence of significant heterogeneity. Sensitivity analysis was used to evaluate the robustness of meta-analyses findings with applying the leave-one-out method after removing one by one included study on the pooled WMDs. Egger regression and Begg's rank correlation tests were applied to detect the potential evidence of publication bias between included studies.

We yielded a total of 8557 records through initial online search in databases. Of these, 1077 were duplicate. After screening based on title and abstract, 304 articles were selected as the candidates for assess according to inclusion and exclusion criteria. Finally, 44 articles (50 studies) were identified to be eligible for current meta-analysis. Figure 1 shows the flowchart of study identification and selection process.

All selected studies contained a total of 7865 (ranging from 13 to1582) patients including 2286

in the severe group and 5579 in the non-severe group. Forty-three of all included articles were conducted in China and one [9] of them was performed in USA.

Most of assessments on laboratory tests among included patients were conducted on admission period/before treatment. The characteristics of included studies are summarized in Table 1 .

Using random-effects model, our meta-analyses indicated a significant decrease in the WMD of 

The pooled finding on cytokines showed a significant increase in the WMD of IL 

We found no significant differences between the pre-and post-sensitivity pooled effect sizes by , [11] the study on TNF-α (WMD= 0.18 pg/mL, 95%CI: -0.03, 0.40), the sensitivity findings showed that there was a significant differences between pre-and post-sensitivity pooled WMD for these outcomes.

Potential publication bias across included studies was examined using the Egger's regression CRP/hs-CRP and ESR have been found to be increased in a vast number of inflammations/infections [46, 51] . In this new pandemic pneumonia, the levels of CRP and ESR significantly increased in severe cases compared to non-severe COVID-19 patients [31, 45] , which greatly coincides with those found in the present systematic review and meta-analysis. In the present study, PCT concentrations were significantly higher in severe/critical patients than in non-severe cases. As it was previously shown, that is, the PCT does not increase with virus infections, it may indicate superimposed bacterial infection for the critically ill patients [47, 52] .

SAA, another important factor capable of improving inflammatory response through activation of chemokine and induction of chemotaxis even at a very low concentration [53] , was found to have elevated circulating levels in severe patients and both were significantly related to COVID-J o u r n a l P r e -p r o o f apoptotic factor [16] . Consequently, these inflammatory-related factors might function as a biomarker to monitor the progression of respiratory diseases.

The higher level of IL-2 in COVID-19 patients is possibly indicative of T cell activation. An important pro-inflammatory cytokine, IL-6 can put an end to the activation of normal T cells, which may be a reason for the presence of lymphopenia. A study carried out by Gong et al. [54] showed that although levels of IL-2R and IL-10 were associated with the severity of the disease, and IL-10) that suppress inflammation; a finding which coincides with that of the present study but differs from SARS-CoV infection [55] . An important anti-viral cytokine generated by CD4+ T cells, CD8+ T cells, NK cells, and macrophages, IFN-γ has been reported to contribute to the cytokines storm in SARS patients [56, 57] . The present study showed that levels of IFN-γ in severely infected cases were higher than those of the non-severe COVID-19 patients, suggesting that IFN-γ may efficiently indicate the status of the disease. Besides, IL-1β and IL-17 were found not to be significantly associated with the COVID-19 severity, which might be due to few numbers of included studies, hence the need for further studies to explain the role that these cytokines play in the progression of the disease. Albeit no significant association was found by some studies between the COVID-19 pneumonia severity and IL-6, IL-10, and TNF-α, this systematic review and meta-analysis indicated that IL-6, IL-10, and TNF-α could be used to assess the severity of COVID-19 and that they might be potential targets for immunotherapy of COVID-19.

The association found between lymphopenia and severity of the COVID-19 implies that, as does SARS-CoV, SARS-CoV-2 might act on lymphocytes, especially T types, hence possibly leading to the depletion of CD4+ T and CD8+ T cells [58] . The exhaustion of CD8+ T cells in severe patients J o u r n a l P r e -p r o o f may reduce their cellular immune response to SARS-CoV-2. The study conducted by Li et al. [59] showed that these multi-functional CD4+ T cells were much frequently seen in patients severely [52, 64] . The elevated levels of NLR found in the present study suggest that the internal environment was seriously disturbed and that the severely infected cases were in a potentially critical condition. Liu et al. [47] revealed that the area under curve (AUC), c-index, sensitivity, and specificity for NLR were at a high level, suggesting that NLR is a reliable index of predicting the incidence of severe illness in an early time. These results point out that the easily accessible tests are potentially easy-to-use, low-cost for early screening and prognosis of the severe and/or critical COVID-19 infected cases.

Whereas COVID-2019 was initially recognized as a pulmonary disease followed by a storm of pro-inflammatory cytokines, resulting in ARDS, MODS, and death [65] , recent evidence indicates that the disease is a systemic disorder affecting many organ systems including kidneys, gastrointestinal tract, liver, nervous system, and skin among others [66] [67] [68] [69] . The mechanism of these systemic effects is not clear yet and many might be related or mediated by the effects of the cytokines and dysregulated immune system [70] .

There are several limitations for this review. As most of the evidence came from China, this lack of evidence from outside China might be a limitation in the way of generalizing our results, particularly with regard to the shortage of costly laboratory tests in the context of nations with low resources. The heterogeneity of the included studies was another limitation, and the need for further studies is greatly felt. One of the main causes might be the poor description of the analytical performance features of the methods applied among the included studies.

Furthermore, aging as a condition related to the inflammation was the basis of the previous studies [62, 71] , showing that severe patients were older than non-severe ones. Since the proinflammatory response is believed to initiate SARS-CoV-2 infection, it is logically possible that the aged cases have an overwhelming inflammatory reaction. Additionally, as the age of some of the patients were not included in some included studies, the comparison of the age differences between the two groups was not possible, which would be another probable limitation. Accordingly, the findings showed that more comprehensive clinical studies are COVID-19 is considered as a global health threat, consequently it is essential that clinicians have access to reliable quick pathogen tests and feasible differential diagnoses based on the clinical descriptions in their first contact with suspected patients. Although it has not been witnessed that pro-inflammatory cytokines and chemokines are directly involved in lung pathology during COVID-19, the changes in laboratory parameters, including the reduced total lymphocytes, lymphocyte subsets, and the elevated NLR, IL-2, IL-4, IL-10, IL-6, and TNF-α, as well as the routine inflammatory-related parameters in infected patients were remarkably associated with the severity of the disease. Likewise, irrespective of the crucial role that hyper-inflammatory responses play in COVID-19 pathogenesis, there could be a protective role for the innate immune system.

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