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Citation: Yafei Zhang, Changtai Wang, Mingfeng Han, Jun Ye, Yong Gao, Zhongping Liu, Tengfei He, Tuantuan Li, Mengyuan Xu, Luping Zhou, Guizhou Zou, Mengji Lu, Zhenhua Zhang. Correction to: Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference [J].VIROLOGICA SINICA, 2020, 35(6) : 885-886.  http://dx.doi.org/10.1007/s12250-020-00293-4

Correction to: Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference

  • Corresponding author: Mengji Lu, mengji.lu@uni-due.de, ORCID: http://orcid.org/0000-0003-4287-9941
    Zhenhua Zhang, zzh1974cn@163.com, ORCID: http://orcid.org/0000-0002-8480-9004
  • The original article can be found online at https://doi.org/10.1007/s12250-020-00273-8.
  • Published Date: 21 September 2020
  • Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%–95.26%) and specificity (83.72%–98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.

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    Correction to: Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference

      Corresponding author: Mengji Lu, mengji.lu@uni-due.de
      Corresponding author: Zhenhua Zhang, zzh1974cn@163.com
    • 1. Department of Infectious Diseases, The Second Hospital of Anhui Medical University, Hefei 230601, China
    • 2. Institute of Clinical Virology, The Second Hospital of Anhui Medical University, Hefei 230601, China
    • 3. Department of Internal Medicine, The Second Hospital of Fuyang, Fuyang 236015, China
    • 4. Institute for Virology, University Hospital of Essen, University of Duisburg-Essen, 45147 Essen, Germany

    Abstract: Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%–95.26%) and specificity (83.72%–98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.

    • Correction to: Virologica Sinica (2020)

      https://doi.org/10.1007/s12250-020-00273-8

      In the original version of Fig. 4I and 4J, the cutoff lines were accidently shifted during figure layout. Figures 4I and 4J are re-drawn and provided below.

      Figure 4.  The RT-PCR detection of SARS-CoV-2 RNAs in 181 sputum and throat swab specimens from 20 patients with conflicting RT-PCR results. 109 paired sputum (A) and 72 throat swab (B) specimens were collected and subjected to SARS-CoV-2 specific RT-PCR assays targeting the ORF1ab and N regions. +: RT-PCR positive; −: RT-PCR negative. Pearson correlation coefficients for the levels of detected SARS-CoV-2 RNA (based on the assays for ORF1ab and N region) and RPP30 RNAs in patient samples were calculated. For sputum specimens: C ORF1ab and N region; D ORF1ab and RPP30; E N region and RPP30. For throat swab specimens: F ORF1ab and N region; G ORF1ab and RPP30; H N region and RPP30. The Ct values of RPP30 RT-PCR were used to analyze the positive and negative results of ORF1ab and N-specific RT-PCR assays with 109 sputum (I) and 72 throat swab (J) specimens. Pos: positive results; Neg: negative results. The negative RT-PCR results of SARS-CoV-2 detection correspond to high Ct values for RT-PCR for RPP30 RNAs. Red lines: cutoffs for ORF1ab RT-PCR; blue lines: cutoffs for N specific RT-PCR. The results were tested for significance by using the Mann–Whitney test (I, J). A P value of < 0.05 is considered as significant.

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