Archives of Academic Emergency Medicine. 2020; 8(1): e43 OR I G I N A L RE S E A RC H Laboratory Parameters in Detection of COVID-19 Patients with Positive RT-PCR; a Diagnostic Accuracy Study Rajab Mardani1, Abbas Ahmadi Vasmehjani2, Fatemeh Zali3, Alireza Gholami4, Seyed Dawood Mousavi Nasab5∗, Hooman Kaghazian5, Mehdi Kaviani6, Nayebali Ahmadi7 † 1. Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran. 2. Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 3. Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Science, Tehran, Iran. 4. WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, Iran. 5. Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran. 6. Expert of Shohada-E Yaft Abad Hospital, Iran University of Medical Sciences, Tehran, Iran. 7. Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Received: February 2020; Accepted: March 2020; Published online: 4 April 2020 Abstract: Introduction: The role of laboratory parameters in screening of COVID-19 cases has not been definitely estab- lished. This study aimed to evaluate the accuracy of laboratory parameters in predicting cases with positive RT-PCR for COVID-19. Methods: This diagnostic accuracy study was conducted on suspected COVID-19 pa- tients, who presented to Behpooyan Clinic Medical center in Tehran (Iran) from 22 February to 14 March, 2020. Patients were divided into two groups based on the results of real time reverse transcriptase-polymerase chain reaction (RT-PCR) for COVID-19, and the accuracy of different laboratory parameters in predicting cases with positive RT-PCR was evaluated using area under the ROC curve (AUC). Results: Two hundred cases with the mean age of 41.3± 14.6 (range: 19-78) years were studied (0.53% male). The result of RT-PCR for COVID-19 was positive in 70 (35%) cases. Patients with positive RT-PCR had significantly higher neutrophil (NEU) count (p = 0.0001), and C-reactive protein (CRP) (p = 0.04), lactate dehydrogenase (LDH) (p = 0.0001), aspartate amino- transferase (AST) (p = 0.001), alanine aminotransferase (ALT) (p = 0.0001), and Urea (p = 0.001) levels in serum. In addition, patients with positive RT-PCR had lower white blood cell (WBC) count (p = 0.0001) and serum albu- min level (p = 0.0001) compared to others. ALT (AUC = 0.879), CRP (AUC = 0.870), NEU (AUC = 0.858), LDH (AUC = 0.835), and Urea (AUC = 0.835) had very good accuracy in predicting cases with positive RT-PCR for COVID-19, respectively. Conclusion: Our findings suggest that level of LDH, CRP, ALT and NEU can be used to predict the result of COVID-19 test. They can help in detection of COVID-19 patients. Keywords: SARS-CoV-2; COVID-19; Biomarkers, Biochemistry; blood cell count; Reverse Transcriptase Polymerase Chain Reaction Cite this article as: Mardani R, Ahmadi Vasmehjani A, Zali F, Gholami A, Mousavi Nasab S D, Kaghazian H, Kaviani M, Ahmadi N. Laboratory Parameters in Detection of COVID-19 Patients with Positive RT-PCR; a Diagnostic Accuracy Study. Arch Acad Emerg Med. 2020; 8(1): e43. ∗Corresponding Author: Seyed Dawood Mousavi Nasab; Department of Re- search and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran. Email: d_mousavinasab@PASTEUR.AC.IR, Tel: +98-0263- 6100990 † Corresponding Author: Nayebali Ahmadi; Proteomics Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Email address: nayebalia@sbmu.ac.ir, Tel: 0098- 021-22714248 1. Introduction Compared to 2002/2003 SARS-CoV and 2012–2014 MERS- CoV epidemics, COVID-19 coronavirus rapidly spread to other parts of the world (185 countries and territories, Last updated: March 21, 2020)(1). In symptomatic patients, the clinical manifestations of the disease usually start after less than a week, consisting of fever (body temperature 37 to 38◦C), cough, nasal conges- tion, and fatigue (2). Pneumonia mostly occurs in the second This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem R. Mardani et al. 2 or third week of a symptomatic infection (3). Comparison of hematological parameters between mild and severe cases of COVID-19 showed significant differences in interleukin-6 (IL-6), D-Dimer, glucose (GLU), thrombin time (TT), fibrino- gen (FIB) and C-reactive protein (CRP) (4). Fan et al. an- alyzed the hematological indices of COVID-19 infected pa- tients between the intensive care unit (ICU) and non-ICU patients. They showed lymphopenia and raised lactate de- hydrogenase (LDH) were associated with higher rate of ICU admissions. Patients who were transferred to the ICU had a lower nadir lymphocyte count, nadir monocyte count and nadir hemoglobin, and higher peak Neutrophil (NEU) Count and peak LDH levels compared to patients who did not re- quire ICU stay (5) . Many patients with MERS-CoV had liver function abnormalities with elevated alanine aminotrans- ferase (ALT), aspartate aminotransferase (AST), and LDH (6). Also laboratory data on SARS have shown that most patients had elevated CRP levels, lymphopenia, leukopenia, and el- evated levels of aminotransferase, LDH and creatine kinase (7). A series of recently published articles have reported the epidemiological and clinical characteristics of patients with COVID-19 disease, but data regarding the laboratory charac- teristics of infected individuals are limited (8-10). This study aimed to evaluate the accuracy of laboratory parameters in predicting cases with positive RT-PCR for COVID-19. 2. Methods 2.1. Study design and setting This diagnostic accuracy study was conducted on suspected COVID-19 patients, who presented to Behpooyan Clinic Medical center in Tehran (Iran) from 22 February to 14 March, 2020. Patients were divided into two groups based on the results of real time reverse transcriptase-polymerase chain reaction (RT-PCR) for COVID-19 and the accuracy of different laboratory parameters in predicting cases with pos- itive RT-PCR was evaluated using area under the ROC curve (AUC). The study protocol was approved by the Ethics Com- mittee of Shahid Beheshti University of Medical Sciences (ethical code: IR.SBMU.RETECH.REC.1399.010). 2.2. Participants Outpatients with suspected COVID-19 having initial respira- tory signs (including sore throat without shortness of breath), fever, cough, muscle ache, and headache were included (1). 2.3. Data gathering Pharyngeal swab samples were collected for COVID-19 test on presentation. Blood samples were collected from each participant and routine blood test including White blood cell count (WBC), Lymphocyte count (LYM), and Neutrophil count (NEU) were performed on the blood samples. Fur- thermore, blood biochemistry parameters such as Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Urea, C-reactive protein (CRP), as well as Albumin and lac- tate dehydrogenase (LDH) were assessed using HITACHI 7600-020 automated biochemistry analyzer. 2.4. Statistical Analysis Data on Urea, WBC, Albumin, AST, ALT, LDH levels were ex- pressed as mean ± standard deviation (SD). Differences in the levels of Urea, CRP, WBC, LYM, NEU, Albumin, AST, ALT and LDH between the RT-PCR positive and negative patients were assessed using student’s t-test. Receiver operating char- acteristic (ROC) curve and AUC were used to analyze the op- timal cut-off for prediction of positive RT-PCR cases. In this study, AUC 0.9 to 1 was defined as excellent accuracy, 0.8 to 0.9 as very good, 0.7 to 0.8 as good, 0.6 to 0.7 as sufficient, 0.5 to 0.6 as bad, and < 0.5 as poor (useless test). 3. Results 3.1. Characteristics of the studied cases Two hundred cases with the mean age of 41.3± 14.6 (range: 19-78) years were studied (0.53% male). 40.2% of cases were in the 30 to 49 years age range. The result of RT-PCR for COVID-19 was positive in 70 (35%) cases and negative in 130 (65%). Groups of patients with positive and negative RT-PCR were similar regarding gender (p = 0.17) and age (p = 0.35) distribution. 3.2. Laboratory parameters Table 1 compares the laboratory parameters of patients with positive and negative RT-PCR. Patients with positive RT-PCR had significantly higher NEU count (p = 0.0001), and CRP (p = 0.04), LDH (p = 0.0001), AST (p = 0.001), ALT (p = 0.0001), and Urea (p = 0.001) levels in serum. In addition, patients with positive RT-PCR had lower WBC count (p = 0.0001) and serum albumin level (p = 0.0001) compared to others. Table 2 and figure 1 show the area under the ROC curve of stud- ied parameters in predicting cases with positive RT-PCR for COVID-19. ALT (AUC = 0.879), CRP (AUC = 0.870), NEU (AUC = 0.858), LDH (AUC = 0.835), and Urea (0.835) had very good accuracy in predicting cases with positive RT-PCR for COVID- 19, respectively. 4. Discussion Based on the findings of this study ALT, CRP, NEU, LDH, and Urea have very good accuracy in predicting cases with posi- tive RT-PCR for COVID-19, respectively. Chen et al., found that LDH had significantly increased in most patients, while albumin had decreased, but ALT and AST showed no significant changes (11). The mentioned val- This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem 3 Archives of Academic Emergency Medicine. 2020; 8(1): e43 Figure 1: Area under the receiver operating characteristic curve of different laboratory parameters in predicting cases with positive RT-PCR for COVID-19. Table 1: Comparing the laboratory parameters between the cases with positive and negative RT-PCR for COVID-19 infection Parameters Total (n=200) RT-PCR for COVID-19 P Positive (n=70) Negative (n=130) WBC (cell/mm3) 5962.8±2127 4043±1002 6894±1982 0.0001 NEU (%) 51.9 60.7 47.8 0.0001 LYM (%) 46.7 37.7 51.8 0.0001 Positive CRPa (%) 37 54 27.6 0.04 AST (IU/L) 28.6±8.6 32.1±8.01 26.8±8.3 0.001 ALT (IU/L) 30±9.1 37.8±7.9 26.2±6.9 0.0001 LDH (U/L) 372.5±115 465.2±100.2 327.6±93.2 0.0001 Urea (mg/dl) 28.6±8.01 34.6±8.6 25.8±5.8 0.001 Albumin (g/dl) 3.5±0.9 2.9±0.8 3.7±0.8 0.0001 a CRP test is qualitative and the indicated number shows the percentage of positive results in each group. Abbreviations: White blood cell count (WBC), Lymphocyte (LYM), Neutrophil (NEU), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), C-reactive protein (CRP), and lactate dehydrogenase (LDH). ues were also reported for patients with MERS-CoV, where el- evated ALT, AST and LDH was observed (6). Another study indicated that 2–11% of patients with COVID-19 had liver co- morbidities and 14–53% of cases had abnormal ALT and AST levels during progression of COVID-19 disease (12). Further- more, Shi et al. studied patients whose COVID-19 diagno- This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem R. Mardani et al. 4 Table 2: The area under the receiver operating characteristic (ROC) curve (AUC) of the studied parameters in predicting cases with positive RT-PCR for COVID-19 Variables Cut-off AUC 95% CI P White blood cell (cells/mm3 ) 0.6 0.075 0.03-0.11 0.09 Neutrophils (%) 0.70 0.858 0.79-0.92 <0.0001 Lymphocyte (%) 0.6 0.112 0.05-0.16 0.12 Positive C-reactive protein (%) 0.70 0.870 0.72-0.88 0.002 Aspartate aminotransferase (IU/L) 0.40 0.716 0.63-0.8 <0.0001 Alanine aminotransferase (IU/L) 0.40 0.879 0.82-0.93 <0.0001 lactate dehydrogenase (U/L) 0.70 0.835 0.76-0.9 <0.0001 Urea (mg/dl) 0.70 0.831 0.76-0.9 <0.0001 Albumin (g/dl) 0.6 0.242 0.15-0.32 0.04 CI: confidence interval. sis was confirmed by computed tomography (CT) scan while in the subclinical phase and found that incidence of AST ab- normality among these patients was significantly lower than those diagnosed after the onset of symptoms (13). Therefore, liver injury is more prevalent in severe cases compared to mild cases of COVID-19. In another report, Yang et al. found no difference in the incidence of abnormal liver function be- tween survivors (30%) and non-survivors (28%) (9). Liver damage in mild cases of COVID-19 is often transient and can return to normal without any special treatment (12). We have found that the number and percentage of WBC, LYM and NEU were significantly different between positive and negative RT-PCR cases for COVID-19/or SARS-CoV-2. In comparison to the normal range, we found low WBC and LYM counts in patients with positive RT-PCR COVID- 19, whereas NEU counts were higher in these patients. In previous reports, low LYM and WBC counts were found in most patients, which is in line with our study (14). Labo- ratory studies showed leucopenia with leukocyte counts of 2.91 ÃŮ 109 cells/L, 70.0% of which were NEU (15). There- fore, our result suggests that NEU might not be affected with SARS-CoV-2 in the initial phase of the disease. It also sug- gests that SARS-CoV-2 might mainly act on lymphocytes, es- pecially T lymphocytes, as does SARS-CoV. Virus particles spread through the respiratory tract and infect other cells, in- ducing series of immune responses, and causing changes in number of peripheral white blood cells such as lymphocytes (11). Some studies suggest that a substantial decrease in the total number of lymphocytes indicates that coronavirus af- fects many immune cells and inhibits cellular immune func- tion (11). Tsui and others reported that high neutrophil count on admission of COVID-19 patients, and elevated LDH level were independent predictors of an adverse clinical outcome (16). In the present study, ROC curve was used to analyze the specificity and sensitivity of different variables in suspected COVID-19 patients. The AUC of laboratory parameters such as ALT, CRP, AST, LDH, and NEU indicated that they could be used to predict the presence of COVID-19 disease, while those of albumin and WBC were below the reference line of ROC curve, indicating that they were poor predictors of the disease. The data is in line with results reported by Wang et al. (17) and Gao et al. (4). In the current study, the AUC of CRP, ALT, LDH, urea and NEU were above 0.80; thus, they are effective and have very good predictive value for predict- ing COVID-19. It seems that, some blood laboratory param- eters could be used in screening cases with positive RT-PCR for COVID-19. Considering the significant difference in laboratory param- eters evaluated in this study between the 2 groups, one can hope to model or predict the results of coronavirus testing based on routine laboratory tests. 5. Limitation The sample size was relatively small. In addition, since this study was conducted on blood laboratory parameters, not every patient was continuously monitored for all clinical manifestations. 6. Conclusion Based on the findings of this study ALT, CRP, NEU, LDH, and Urea have very good accuracy in predicting cases with posi- tive RT-PCR for COVID-19, respectively. 7. Declarations 7.1. Acknowledgements The authors thank all their colleagues who co-operated in this investigation. 7.2. Author contribution S.D.M.N and A.A.V conceived and designed experiments. M.K. and R.M performed the experiments. A.R.G, N.A, F.Z and H.K analyzed the data. S.D.M.N. A.A.V and N.A wrote the paper. N.A revised and edited the paper. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem 5 Archives of Academic Emergency Medicine. 2020; 8(1): e43 Authors ORCIDs Rajab Mardani: 0000-0001-7730-3040 Abbas Ahmadi Vasmehjani: 0000-0001-8079-7352 Fatemeh Zali: 0000-0001-9098-4418 Nayebali Ahmadi: 0000-0002-8870-7267 Hooman Kaghazian: 0000-0003-4301-3381 Alireza Gholami: 0000-0003-0706-2821 Mehdi kaviani: 0000-0002-9400-3207 Seyed Dawood Mousavi Nasab: 0000-0002-4831-3108 7.3. Funding/Support This study was supported by the Proteomics Research Center of Shahid Beheshti University of Medical Sciences. 7.4. Conflict of interest The authors have no conflicts of interest. References 1. Organization WH. Clinical management of severe acute respiratory infection (SARI) when COVID-19 disease is suspected: interim guidance, 13 March 2020. World Health Organization, 2020. 2. Guan W-j, Ni Z-y, Hu Y, Liang W-h, Ou C-q, He J-x, et al. Clinical characteristics of coronavirus disease 2019 in China. New England Journal of Medicine. 2020. 3. 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The Journal of Infectious Diseases. 2020. This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 3.0 License (CC BY-NC 3.0). Downloaded from: http://journals.sbmu.ac.ir/aaem Introduction Methods Results Discussion Limitation Conclusion Declarations References