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      False Negative RT-PCR and False Positive Antibody Tests ——Concern and Solutions in the Diagnosis of COVID-19

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          Abstract

          Sir, We read with interest that antibody testing using a rapid immunochromatographic assay is reliable in the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. 1 However, the accuracy of antibody testing and RT-PCR does not meet the need for a large number of screening tests. False negative RT-PCR and false positive antibody tests are a concern. Coronavirus disease 2019 (COVID-19), which is caused by SARS-Cov-2, was first detected at the end of 2019, and was named by the World Health Organization on January 12, 2020. COVID-19 is now a pandemic. It took only 25 days for newly confirmed cases to decrease to zero in Beijing in June, which revealed that timely discovery, accurate diagnosis, early isolation and treatment of COVID-19 are the most effective measures. Suspected cases were judged by epidemiological history and clinical manifestations. Confirmed cases were diagnosed by real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) which identified the new coronavirus nucleic acid and serum IgM/IgG antibody tests. Infected persons with false negative RT-PCR results may not be isolated and can infect others. 2 False positive results can cause panic among patients and doctors. Furthermore, unnecessary crowd isolation wastes human and material resources. Different types of clinical specimens and thermal inactivation may cause false negative RT-PCR results. Nucleic acid detection has the limitation of a low positive rate in different types of clinical specimens. Wenling Wang's research revealed that bronchoalveolar lavage fluid specimens showed the highest positive rates (14 of 15; 93%), followed by sputum (72 of 104; 72%), nasal swabs (5 of 8; 63%), fibrobronchoscope brush biopsy (6 of 13; 46%), pharyngeal swabs (126 of 398; 32%), feces (44 of 153; 29%), and blood (3 of 307; 1%). 3 We recommend that upper respiratory tract specimens be collected in the acute phase and lower respiratory tract specimens or feces samples be collected in the non-acute phase. Based on the knowledge of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV, thermal inactivation at 56°C was recommended to inactivate SARS-CoV-2 before nucleic acid testing. However, Pan Y's research demonstrated that thermal inactivation affected the efficiency of RT-PCR for SARS-CoV-2 detection and chemical inactivators, such as guanidinium-based lysis, are suggested. His study showed that approximately half of the weakly positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel test. 4 At the initial stage of the SARS-CoV-2 epidemic in February 2020, we observed 57 suspected COVID-19 infected patients. Pharyngeal swabs were tested for nucleic acid and serum samples were obtained for 2019-nCOV IgM and IgG tests. The positive rate of COVID-19 nucleic acid was 42.10%. The positive detection rate of combined 2019-nCOV IgM and IgG for patients with COVID-19 negative and positive nucleic acid tests was 72.73% and 87.50%, respectively. 5 These data demonstrated that the detection of novel coronavirus antibodies is an important supplementary method for the diagnosis of COVID-19. In China's trial version 7 of the diagnosis and treatment guideline for the novel coronavirus disease (COVID-19), serum novel coronavirus specific IgM antibody and IgG antibody were regarded as criteria for confirming COVID-19 in patients on March 3, 2020. A study involving 1,779 patients in Iceland, showed that 91.1% of those who recovered from SARS-COV-2 infection tested seropositive. 6 It was found that the antibody test may be affected by other factors such as rheumatoid factor (RF) in the serum. 7 Rheumatoid patients often have different concentrations of RF, and RF has five types of immunoglobulins including IgM, IgG, IgD, IgA and IgE. A high concentration of RF-IgM may interfere with novel coronavirus IgM/IgG antibody detection. The effects of RF concentration from 16.1 IU/mL to 539.7 IU/mL on the detection of 2019-nCoV IgM and IgG tests were observed. It was found that RF may lead to false results when the serum RF level was higher than 231.7 IU/mL. The false positive antibody results could be eliminated after five times dilution with normal human serum, when the RF level was lower than 10 IU/mL. It was not eliminated after five times dilution with physiological saline[Fig. 1 ]. We also identified five patients with false antibody results, who had nasopharyngeal carcinoma, colon cancer, duodenal carcinoma, diabetes, and diffuse bronchitis, respectively. Serum RF level in these patients was lower than 100 IU/mL. The false positive antibody results could also be eliminated after 5 times dilution with normal human serum. Thus, further studies are needed to investigate the false results of this test. Figure 1 Influence and elimination experiment resulting in a false positive test result, which was affected by serum rheumatoid factor levels. Eleven different serum concentrations of rheumatoid factor were tested for 2019-nCoV IgM and IgG. From left to right, RF was 16.1, 85.0, 133.0, 187.5, 231.7, 284.3, 331.0, 390.5, 450.0, 490.6, and 539.7 (IU/mL). A is the original result, B and C are the results diluted five times with normal human serum and physiological saline, respectively. Figure 1 We believe that no diagnostic technique has 100% sensitivity and specificity. Although the RT-PCR test has become the standard method for the diagnosis of SARS-CoV-2 infection, false-negative rates have been reported. For the serological antibody test, the detection time needs to consider the window period. Moreover, several factors should be considered when diagnosing COVID-19, including epidemiology, history of exposure and clinical symptoms, such as fever or respiratory disease. Therefore, the combination of serum IgM/IgG antibody detection, the nucleic acid test, CT scan and clinical features improves the accuracy of COVID-19 diagnosis. Declaration of Competing Interest The authors declare that they have no conflict of interest.

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          Most cited references6

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          Detection of SARS-CoV-2 in Different Types of Clinical Specimens

          This study describes results of PCR and viral RNA testing for SARS-CoV-2 in bronchoalveolar fluid, sputum, feces, blood, and urine specimens from patients with COVID-19 infection in China to identify possible means of non-respiratory transmission.
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            Humoral Immune Response to SARS-CoV-2 in Iceland

            Abstract Background Little is known about the nature and durability of the humoral immune response to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods We measured antibodies in serum samples from 30,576 persons in Iceland, using six assays (including two pan-immunoglobulin [pan-Ig] assays), and we determined that the appropriate measure of seropositivity was a positive result with both pan-Ig assays. We tested 2102 samples collected from 1237 persons up to 4 months after diagnosis by a quantitative polymerase-chain-reaction (qPCR) assay. We measured antibodies in 4222 quarantined persons who had been exposed to SARS-CoV-2 and in 23,452 persons not known to have been exposed. Results Of the 1797 persons who had recovered from SARS-CoV-2 infection, 1107 of the 1215 who were tested (91.1%) were seropositive; antiviral antibody titers assayed by two pan-Ig assays increased during 2 months after diagnosis by qPCR and remained on a plateau for the remainder of the study. Of quarantined persons, 2.3% were seropositive; of those with unknown exposure, 0.3% were positive. We estimate that 0.9% of Icelanders were infected with SARS-CoV-2 and that the infection was fatal in 0.3%. We also estimate that 56% of all SARS-CoV-2 infections in Iceland had been diagnosed with qPCR, 14% had occurred in quarantined persons who had not been tested with qPCR (or who had not received a positive result, if tested), and 30% had occurred in persons outside quarantine and not tested with qPCR. Conclusions Our results indicate that antiviral antibodies against SARS-CoV-2 did not decline within 4 months after diagnosis. We estimate that the risk of death from infection was 0.3% and that 44% of persons infected with SARS-CoV-2 in Iceland were not diagnosed by qPCR.
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              False Negative Tests for SARS-CoV-2 Infection — Challenges and Implications

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                Author and article information

                Journal
                J Infect
                J Infect
                The Journal of Infection
                Published by Elsevier Ltd on behalf of The British Infection Association.
                0163-4453
                1532-2742
                8 October 2020
                8 October 2020
                Affiliations
                [0001]Southern Medical University, Department of Clinical Laboratory Medicine Center, Shenzhen Hospital, 1333 Xinhu Road, Baoan District, Shenzhen, Guangdong, China
                Author notes
                [* ]Corresponding author.
                Article
                S0163-4453(20)30649-6
                10.1016/j.jinf.2020.10.007
                7543921
                5f4b9c76-a3c2-4742-b1d6-5c89f0f74e65
                © 2020 Published by Elsevier Ltd on behalf of The British Infection Association.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                Categories
                Letter to the Editor

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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