key: cord-0888891-65plab8b
authors: Jiang, Guanmin; Ren, Xiaoshuai; Liu, Yan; Chen, Hongtao; Liu, Wei; Guo, Zhaowang; Zhang, Yaqin; Chen, Chaoqun; Zhou, Jianhui; Xiao, Qiang; Shan, Hong
title: Application and optimization of RT-PCR in diagnosis of SARS-CoV-2 infection
date: 2020-02-27
journal: nan
DOI: 10.1101/2020.02.25.20027755
sha: e046a06f262d74d025272ad697d358c4afb5d9bd
doc_id: 888891
cord_uid: 65plab8b

Background: Coronavirus Disease 2019 (COVID-19) caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global threat to public health. Aiming to construct an efficient screening pattern, we comprehensively evaluated the performances of RT-PCR and chest CT in diagnosing COVID-19. Methods: The records including demographics, RT-PCR, and CT from 87 confirmed COVID-19 cases and 481 exclusion cases were collected. The diagnostic accuracy of the pharyngeal swab RT-PCR, CT, combination with the second pharyngeal swab RT-PCR or with CT were evaluated individually. Besides, all the stool RT-PCR results were plotted by time to explore the value of stool RT-PCR. Findings: Combination of RT-PCR and CT has the higher sensitivity (91.9%,79/86) than RT-PCR alone (78.2%，68/87) or CT alone (66.7%, 54 of 81) or combination of two RT-PCR tests (86.2%,75/87). There was good agreement between RT-PCR and CT (kappa-value, 0.430). In 34 COVID-19 cases with inconsistent results, 94.1% (n=32) are mild infection, 62.5% of which (20/32) showed positive RT-PCR. 46.7% (35/75) COVID-19 patients had at least one positive stool during the course. Two cases had positive stool earlier than the pharyngeal swabs. Importantly, one patient had consecutive positive stool but negative pharyngeal swabs. Interpretation: Combination of RT-PCR and CT with the highest sensitivity is an optimal pattern to screen COVID-19. RT-PCR is superior to CT in diagnosing mild infections. Stool RT-PCR should be considered as an item for improving discovery rate and hospital discharge. This study shed light for optimizing scheme of screening and monitoring of SARS-CoV-2 infection.

In early December 2019, the first pneumonia cases of unknown origins were identified in Wuhan city, Hubei province, China 1 . On Jan 7, a novel coronavirus was discovered using high-throughput sequencing in the throat swab sample of a patient , and is currently named SARS-CoV-2(previously known as 2019-nCoV)on February 11, 2020 by ICTV 2,3 . The initial defined cases of COVID-19, were epidemiologically linked to the human seafood market in Wuhan, Although later more and more COVID-19 were found without exposure the market but with a history to Wuhan or contact with the patient of COVID-19 pneumonia confirmed 2,4,5 . Current epidemiologic data indicate the Fever, cough and dyspnea were the most common symptoms in patients with COVID-19 pneumonia. A manifestation similar of those of two other disease caused by coronaviruses, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) [18] [19] [20] . CT is an important method in the diagnosis of lung lesions, and the radiological changes in the lungs of COVID-19 patients has been characterized 21 . Zhong et al. reported that of 840 COVID-19 patients who underwent CT on admission, around 76.4% manifested abnormal CT imaging features and usually exhibited typical radiological finding of the ground-glass opacity (50%) or bilateral patchy shadowing (46%) 22 .

Based on the "Diagnosis and Treatment Guideline for New Coronavirus Pneumonia (the fifth edition), China", CT scan were used as the clinical diagnostic criteria for COVID-19 pneumonia, but strictly limited in Hubei Province 23 . However, the specificity of chest CT is relatively low，alone could not distinguish the SARS-CoV-2 infection from other pathogens well.

SARS-CoV-2 causes extensively outbreak in cold winter. In this season, many other pathogens causing pneumonia also become prevalent, even including many viral agent. The infectious diseases share some common characteristics in signs, symptoms and laboratory findings. Therefor it is difficult to differentiate COVID-19 suffers from other pneumonia patients purely depending to the manifestation or routine examination. Therefore , an precision screening scheme is urgent to be employed. High sensitive test is pivotal to avoiding secondary transmission by missed diagnosed cases.

Meanwhile, the positive predictive value also should be counted, for a number of false positive would bring out not only occupation and cost of healthcare resource, but also increasing infection risk of suspected cases isolated in hospital. In this study, we performed a retrospective study in the 568 cases and compare the efficacy of RT-PCR and CT diagnostic approaches in COVID-19 diagnosis, and to provide evidence for future strategic diagnosis in regions outside Hubei Province.

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For this retrospective, single center study, we recruited 584 patients from Jan 17 to Feb 11, 2020, at The Fifth Affiliated Hospital of Sun Yat-sen University in Zhuhai, China, which is a designated infectious hospital. During this period, RT-PCR and chest CT was performed for consecutive patients including the local residents of Wuhan, outside of Wuhan did have a recent travel to Wuhan or contact with people with fever or respiratory symptoms from Wuhan, or had fever or acute respiratory symptoms of unknown cause. Of the 16 patients recruited as of Feb 11, had a suspected diagnosis and were therefore excluded in this study. 87 patients, who were diagnosed as having COVID-19 and 481 patients exclusion COVID-19 according to WHO interim guidance, were enrolled in this study. The performances of the first RT-PCR detection in pharyngeal swabs and chest CT were evaluated by sensitivity, specificity, youden's index et al. Then the performances of combination of the second RT-PCR, or chest CT were also calculated. Agreement between the two method was analyzed using

McNemar Chi-squared test. Finally the all RT-PCR results from pharyngeal and stool were plotted by time to explore the value of stool nucleic detection (Fig 1) . The severity of COVID-19 pneumonia was defined based on the international guidelines for community-acquired pneumonia 24 . Laboratory and CT characteristics data were obtained with standard data collection forms from electronic medical records.

The study was approved by The Fifth Affiliated Hospital of Sun Yat-sen University Ethics Committee and written informed consent was obtained from patients involved before enrolment when data were collected retrospectively.

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The SARS-Cov-2 laboratory test assays were based on the previous WHO recommendation. The upper respiratory tract specimens (pharyngeal and nasal swabs) and stool were obtained from all the cases. Ensure each specimen collected has the name, gender and age of the patient as well as a serial number; any abnormality in the specimen should be noted.

RNA was extracted and tested by real-time RT-PCR with SARS-Cov-2 specific primers and probes according to instruction of Kit. The real-time RT-PCR was carried out in biosafety level 2 facilities at the clinic laboratory. If two targets (RdRp+, E or N +) tested positive by specific RT-PCR, the patients would be considered to be laboratory-confirmed.

Negative: no Ct value or Ct 40.

Positive: a Ct value < 37.

A Ct value between 37-40 is indeterminate. It is required confirmation by repeating. If, when repeated, the Ct value is < 37 the sample is positive, otherwise, it is negative. . CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20027755 doi: medRxiv preprint 1 1 81 of which were COVID-19 patients. We looked through the case data retrospectively and found that 5 patients without CT scan were admitted to hospital complaining of epidemiology contact. Although they were eventually diagnosed, they had only mild symptoms of upper respiratory tract infection on admission and in the course of infection, and no CT scan had been performed.

In the beginning of the SARS-CoV-2 outbreak, RT-PCR for pharyngeal and CT scan is the main Besides that, we also analyze the agreement of the two methods (The cases with uncertain CT results were not involved in statistical analysis due to statistical limitations). As is shown in Table3, there was statistically significance between the two methods (p<0.001). Moreover, there was statistically differences in the diagnosis of non-COVID-19 patients(p<0.001), but not in COVID-19

patients (p=0.734). The agreement was good to fair agreement (kappa value, 0.430) , and the adjusted agreement was 72.8% in all patients.

. CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20027755 doi: medRxiv preprint Given that the time on receiving detection can cause influences on sensitivity, the time differences between RT-PCR for pharyngeal and CT scan were compared. The average time of nucleic acid screening was earlier than that of CT scan statistically (-0.7390days, -3625~1.674) ( Table 4) It is notable that both of the methods acquired the sensitivity less than 80% in screening SARS-CoV-22 infections, which is not ideal enough for the diagnosis of infectious diseases with severe consequences. To develop more appropriate detection scheme, the performances of combination . CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20027755 doi: medRxiv preprint 1 . CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20027755 doi: medRxiv preprint The day differences between the first RT-PCR detection and CT scan were performed statistical analysis.

*One-sample t test were used to test statistical significance between the mean of group and zero at the p <0.05 level.

# Independent sample t test were used to test statistical significance of the day differences between non-COVID-19group and COVID-19 group.

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is the (which was not peer-reviewed) The copyright holder for this preprint . CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20027755 doi: medRxiv preprint . CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.02.25.20027755 doi: medRxiv preprint

The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health -The latest 2019 novel coronavirus outbreak in Wuhan, China. International journal of infectious diseases : IJID : official publication of the International Society for