Sample-to-Answer and Routine Real-Time RT-PCR

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading all over the world and has caused millions of deaths. Several sample-to-answer platforms, including Cepheid Xpert Xpress SARS-CoV-2 (Xpert Xpress), have received emergency use authorization for SARS-CoV-2 nucleic acid detection as a point of care test in the United States. However, their application niche is unclear when compared with real-time RT-PCR assays cleared by the National Medical Products Administration in China. In this study, the clinical performance, sensitivity, and workflow of Xpert Xpress and two real-time RT-PCR kits (BioGerm kit and Sansure kit) were evaluated by the specimens from 86 symptomatic patients. The positive percent agreement of Xpert Xpress was 100% compared with 96.15% for the BioGerm kit and 90% for the Sansure kit. The negative percent agreement was 100% for all three assays. The limit of detection is 100 copies/mL for Xpert Xpress and 500 copies/mL for the BioGerm kit and Sansure kit. By serially diluting five positive specimens, the Xpert Xpress had better detection capability. In the workflow and throughput analysis, the turnaround time was 51 minutes for Xpert Xpress, 150 minutes for the BioGerm kit, and 210 minutes for the Sansure kit. This study provides some indication for diagnosis methods selection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading all over the world and has caused millions of deaths. Several sample-to-answer platforms, including Cepheid Xpert Xpress SARS-CoV-2 (Xpert Xpress), have received emergency use authorization for SARS-CoV-2 nucleic acid detection as a point of care test in the United States. However, their application niche is unclear when compared with real-time RT-PCR assays cleared by the National Medical Products Administration in China. In this study, the clinical performance, sensitivity, and workflow of Xpert Xpress and two real-time RT-PCR kits (BioGerm kit and Sansure kit) were evaluated by the specimens from 86 symptomatic patients. The positive percent agreement of Xpert Xpress was 100% compared with 96.15% for the BioGerm kit and 90% for the Sansure kit. The negative percent agreement was 100% for all three assays. The limit of detection is 100 copies/mL for Xpert Xpress and 500 copies/mL for the BioGerm kit and Sansure kit. By serially diluting five positive specimens, the Xpert Xpress had better detection capability. In the workflow and throughput analysis, the turnaround time was 51 minutes for Xpert Xpress, 150 minutes for the BioGerm kit, and 210 minutes for the Sansure kit. This study provides some indication for diagnosis methods selection. The novel coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a pandemic all over the world.1Wang C. Horby P.W. Hayden F.G. Gao G.F. A novel coronavirus outbreak of global health concern.Lancet. 2020; 395: 470-473Abstract Full Text Full Text PDF PubMed Scopus (5090) Google Scholar, 2Zhu N. Zhang D. Wang W. Li X. Yang B. Song J. Zhao X. Huang B. Shi W. Lu R. Niu P. Zhan F. Ma X. Wang D. Xu W. Wu G. Gao G.F. Tan W. A novel coronavirus from patients with pneumonia in China, 2019.N Engl J Med. 2020; 382: 727-733Crossref PubMed Scopus (18849) Google Scholar, 3The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.Nat Microbiol. 2020; 5: 536-544Crossref PubMed Scopus (5058) Google Scholar As of January 31, 2021, >101 million infection cases had been confirmed, including more than 2.19 million deaths (World Health Organization, https://covid19.who.int, last accessed January 31, 2021). Until now, SARS-CoV-2, which is a contagion with a high rate of transmission from human to human, has been the seventh coronavirus known to infect humans.4Loeffelholz M.J. Tang Y.W. Laboratory diagnosis of emerging human coronavirus infections - the state of the art.Emerg Microbes Infect. 2020; 9: 747-756Crossref PubMed Scopus (555) Google Scholar Individuals who contract SARS-CoV-2 can experience mild to severe respiratory tract illness, and symptoms including fever, cough, myalgia or fatigue, sputum production, and headache.5Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. Xie X. Yin W. Li H. Liu M. Xiao Y. Gao H. Guo L. Xie J. Wang G. Jiang R. Gao Z. Jin Q. Wang J. Cao B. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395: 497-506Abstract Full Text Full Text PDF PubMed Scopus (33262) Google Scholar In addition, older individuals and those with underlying medical problems (such as cardiovascular disease, diabetes, chronic respiratory disease, and cancer) are more likely to develop serious illness (World Health Organization, https://www.who.int/health-topics/coronavirus#tab=tab_1, last accessed January 31, 2021). Given that SARS-CoV-2 has a high rate of transmission and can be fatal, the foremost priority to facilitate public health intervention and to contain COVID-19 is reliable laboratory diagnosis. Real-time RT-PCR is one of the routine methods for diagnosis of pathogen in contagion. The detection of pathogen by real-time RT-PCR was performed in the traditional molecular laboratory by staffs with special technical training. Recently, several sample-to-answer platforms were approved to detect SARS-CoV-2 by the US Food and Drug Administration (FDA), such as the Xpert Xpress SARS-CoV-2 assay (Cepheid, Sunnyvale, CA), ID NOW COVID-19 assay (Abbott Laboratories, Chicago, IL), and ePlex SARS-CoV-2 assay (GenMark, Carlsbad, CA).6Zhen W. Smith E. Manji R. Schron D. Berry G.J. Clinical evaluation of three sample-to-answer platforms for the detection of SARS-CoV-2.J Clin Microbiol. 2020; 58: e00783-20Crossref PubMed Scopus (182) Google Scholar However, comparisons between sample-to-answer assays and routine real-time RT-PCR are lacking. Because of the good performance of the Xpert Xpress SARS-CoV-2 platform,6Zhen W. Smith E. Manji R. Schron D. Berry G.J. Clinical evaluation of three sample-to-answer platforms for the detection of SARS-CoV-2.J Clin Microbiol. 2020; 58: e00783-20Crossref PubMed Scopus (182) Google Scholar this assay was compared with two traditional real-time RT-PCR kits that have been approved to detect SARS-CoV-2 with permission of the National Medical Products Administration (NMPA): the Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit (PCR-fluorescence probing) (BioGerm Medical Biotechnology Co. Ltd., Shanghai, China) (BioGerm kit) and the 2019-nCoV Nucleic Acid Diagnostic Kit (PCR-fluorescence probing) (Sansure Biotech Inc., Changsha, China) (Sansure kit). In this study, the analytical and clinical performance and the workflow of the FDA-cleared Xpert Xpress SARS-CoV-2 assay platform with two NMPA-cleared routine real-time RT-PCR kits were compared. Nylon swabs and viral transport tubes with 3 mL of preservation medium were prepared. One pharyngeal swab and two nasal swabs (nasopharyngeal swabs) were collected from each symptomatic patient from the fever clinic. The three swabs were placed into a viral transport tube. Swabs were broken at the indicated break line and the specimen collection tube capped tightly. Then specimens were transported to the clinical laboratory. Before nucleic acid detection, the SARS-CoV-2 was inactivated by 56°C for 30 minutes. After routine detection, samples were aliquoted and stored at −80°C immediately. A number of 34 fresh specimens, including 6 positive and 28 negative specimens, were detected parallelly by Xpert Xpress and the BioGerm kit. A total of 86 specimens, including 26 positive specimens and 60 negative specimens, detected during March and October 2020 were selected for this study. The 26 positive specimens were from the patients, who were diagnosed by clinical symptom, computed tomography, SARS-CoV-2 nucleic acid detection, or specific antibody detection according to the Seventh Revised Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment Guidance (National Health Commission, http://www.nhc.gov.cn/yzygj/s7653p/202003/46c9294a7dfe4cef80dc7f5912eb1989.shtml, last accessed January 31, 2021) and were then transferred and admitted to Shanghai Public Health Clinic Center for quarantine and advanced treatment. Retrospective samples were removed from the freezer at −80°C, thawed, aliquoted, and then tested immediately by Xpert Xpress, the BioGerm kit, and the Sansure kit in parallel. RNA was extracted from nasopharyngeal swab specimens using combined nucleic acid extraction and purification kits according to the manufacturer's instruction. A total of 200 μL of nasopharyngeal swab specimen was added into one well of a prepared plate. Then the plate was loaded onto the fast nucleic acid extraction machinery. Nucleic acid was eluted with 100 μL of elution buffer. The real-time RT-PCR for SARS-CoV-2 detection were performed by using a 2019-nCoV Nucleic Acid Detection Kit (PCR-fluorescence probing) (BioGerm Medical Biotechnology Co. Ltd.) with permission of the NMPA. Each real-time RT-PCR reaction had a final volume of 25 μL, including 5 μL of nucleic acid purification product and 20 μL of real-time RT-PCR mixture. The BioGerm kit detects the ORF1ab gene and the N gene. The result was determined as positive when the Ct values of both the ORF1ab and N genes were ≤38; otherwise, a negative result was determined when the Ct values of both the ORF1ab and N genes were >38. An ORF1ab or N gene Ct value ≤38 was considered as a presumptive positive result and retesting was required; if the ORF1ab or N gene Ct value was ≤38 for the second time, the result was considered positive. A total of 200 μL of the nasopharyngeal swab specimen was prepared for RNA extraction for each test. RNA extraction was operated using combined nucleic acid extraction kits (catalog number S1006) on Natch CS machinery. The nucleic acid was eluted with 100 μL of elution buffer. The real-time RT-PCR for SARS-CoV-2 detection was performed by using 2019-nCoV Nucleic Acid Diagnostic Kit (PCR-fluorescence probing) (Sansure Biotech Inc.) with permission of the NMPA. Each real-time RT-PCR reaction had final volume of 50 μL, including 20 μL of nucleic acid purification product and 30 μL of the real-time RT-PCR mixture. The Sansure kit detects the ORF1ab and N genes. The result was considered positive when the Ct values of both the ORF1ab and N genes were ≤40; otherwise, when the Ct values of both the ORF1ab and N genes were >40, the result was considered negative. For each testing, 300 μL of the nasopharyngeal swab specimen was added into the sample chamber in the cartridge using a transfer pipette. Then the lid was closed and the cartridge loaded onto the GeneXpert machinery for automated sample processing and in real-time viral RNA detection. If the SARS-CoV-2 signal for the N2 nucleic acid target or signals for both nucleic acid targets (N2 and E) had Ct values within the valid range and end points above the minimum setting, this was considered a positive result. If the SARS-CoV-2 signal for only the E nucleic acid target had a Ct within the valid range and an end point above the minimum setting, the result was considered presumptive positive, and the testing was repeated. If the SARS-CoV-2 signals for two nucleic acid targets (N2 and E) do not have a Ct within the valid range and an end point above the minimum setting, the result was considered negative. If the Ct values of both N2 and E were <45, the result was considered positive. If the Ct value of a single target was <45, the result was considered positive for N2 and presumptive for E. The SARS-CoV-2 RNA standard substrate [catalog number GBW(E)091099; Chinese National Institute of Metrology, Beijing China] was used for a limit of detection (LOD) study. The concentration was 6.89 × 105 copies/mL for ORF1ab, 1.36 × 106 copies/mL for N, and 8.04 × 105 copies/mL for E. ORF1ab and N were diluted to designated concentrations and detected. At the designated concentration, five replicates were tested for each gene. Five specimens with high Ct value (ORF1ab: 18.50 to 23.56; N: 20.23 to 24.09 by the BioGerm Kit) were selected for serial dilution using a mixture of negative samples. The dilution ratio were 1:101, 1:102, 1:103, 1:104, 1:105, and 1:106 for specimens 1 to 5, respectively. The diluted samples were aliquoted and tested immediately by Xpert Xpress, the BioGerm kit, and the Sansure kit in parallel. Positive rates were calculated and used to evaluate the sensitivity of the three testing methods. Anti-SARS-CoV-2 antibodies were detected by a 2019-nCoV Ab Test (Colloidal Gold; Innovita Biotechnology Co., Ltd., Tangshan, China). It is an immunochromatographic assay for rapid, qualitative detection of anti–SARS-CoV-2 IgM and IgG antibodies in human serum. A total of 10 μL of plasma specimen was transferred to the sample IgM-detecting and IgG-detecting wells, and then 2 to 3 drops (80 μL) of buffer solution were added to each well, respectively. After 15 minutes, the results were read. Reading results after 20 minutes was forbidden. The reference standard was established by the Seventh Revised Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment Guidance. The positive percent agreement and the negative percent agreement were calculated using Microsoft Office Excel 2007 software (Microsoft Inc., Redmond, WA), and κ values with 95% CIs were calculated by SPSS software version 20.0 (SPSS Inc., Chicago, IL). Clinical testing was executed on 86 specimens, including 60 negative specimens and 26 positive specimens. The 26 COVID-2019 cases were confirmed by clinical symptom, computed tomography, SARS-CoV-2 nucleic acid detection, or specific antibody detection according to the Seventh Revised Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment Guidance. These patients with COVID-2019 were transferred and admitted to Shanghai Public Health Clinic Center for quarantine and advanced treatment. The Xpert Xpress detected 26 SARS-CoV-2–positive cases, and the BioGerm kit detected 25 positive cases. Among the positive cases, the Sansure kit tested 20, and 18 cases tested positive (Table 1). The positive percent agreement of Xpert Xpress was 100%, followed by 96.15% for the BioGerm kit and 90% for the Sansure kit. The negative percent agreement was 100% for all three methods. The Xpert Xpress detected one SARS-CoV-2–positive case that was not detected by the BioGerm kit or Sansure kit. This specimen was from a patient with COVID-2019 whose anti-–SARS-CoV-2 antibody IgM test result was negative but whose IgG test result was positive. There was another specimen from which SARS-CoV-2 could be detected by Xpert Xpress and the BioGerm kit but not by the Sansure kit. This specimen was from a patient with COVID-2019 whose anti–SARS-CoV-2 antibody IgM test result was negative and whose IgG test result was weakly positive (Table 2). It was suggested that those two patients with COVID-2019 were convalescents.Table 1Clinical Performance Comparison of Three AssaysAssayClinical guidelinesκ∗Strong (0.75 to 1.00), moderate (0.40 to 0.75), or weak (0.0 0 to 0.40).PPA, %NPA, %PositiveNegativeBioGerm kit Positive2500.9796.15100 Negative160Sansure kit Positive1800.9290100 Negative232Xpert Xpress Positive2601.00100100 Negative060∗ Strong (0.75 to 1.00), moderate (0.40 to 0.75), or weak (0.0 0 to 0.40). Open table in a new tab Table 2Details of Discordant SamplesClinical guidelinesAntibody test resultCt valueBioGerm kitSansure kitXpert XpressIgMIgGORF1abNResultORF1abNResultN2EResultPositiveNegativePositive>40>40Negative>4542.8Negative42.6>45PositivePositiveNegativePositive37.737.9Positive>4541.9Negative39.3>45Positive Open table in a new tab The LOD was determined using SARS-CoV-2 RNA from the Chinese National Institute of Metrology. Before testing, the SARS-CoV-2 RNA was diluted in a negative clinical specimen. The positivity rate observed was ≥100% at a lowest SARS-CoV-2 concentration of 100 copies/mL for Xpert Xpress and 500 copies/mL for the BioGerm and Sansure kits (Table 3). Therefore, the LOD is 100 copies/mL for Xpert Xpress and 500 copies/mL for the BioGerm and Sansure kits.Table 3Limits of Detection Evaluated by the Standard SubstrateConcent-rationXpert XpressBioGerm kitSansure kitN2 (Ct ≤ 45)E (Ct ≤ 45)Orf1ab (Ct ≤ 38)N (Ct ≤ 38)Orf1ab (Ct ≤ 40)N (Ct ≤ 40)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)500 copies/mL34.615/5 (100)36.855/5 (100)35.645/5 (100)38.555/5 (100)34.9336.1935.0437.7333.3835.9336.5938.3835.3235.5636.5438.1035.4336.3736.6238.32200 copies/mL0.000/5 (0)38.623/5 (60)36.764/5 (80)39.265/5 (100)38.9137.3236.6638.7639.010.0036.6338.470.0037.490.0039.7939.1637.3438.3739.22100 copies/mL42.105/5 (100)36.805/5 (100)0.000/5 (0)0.001/5 (20)0.000/5 (0)40.922/5 (40)42.4039.8038.9137.270.0040.8340.5038.1039.000.000.0039.9341.1039.000.0038.320.0042.2042.6041.7039.000.000.0039.59 Open table in a new tab To evaluate the sensitivity of the three methods to the clinical specimens, five positive specimens with high Ct values (ORF1ab: 18.50 to 23.56; N: 20.23 to 24.09 by the BioGerm kit) were diluted into serial dilutions with mixture of negative samples. The dilution ratios were 1:10ˆ0, 1:101, 1:102, 1:103, 1:104, 1:105, and 1:106 for samples 1 to 5, respectively. The diluted samples were tested by Xpert Xpress, the BioGerm kit, and the Sansure kit in parallel. The result indicate that at a dilution ratio <1:104,Xpert Xpress, the BioGerm kit, and the Sansure kit had almost equal detective ability, but when the dilution ratio was >1:105, the Xpert Xpress had better detection capability (Table 4).Table 4Detection of Serial Dilution Samples Derived from Five Positive Specimens by Three AssaysDilution rateXpert XpressBioGerm kitSansure kitN2 (Ct ≤ 45)E (Ct ≤ 45)Orf1ab (Ct ≤ 38)N (Ct ≤ 38)Orf1ab (Ct ≤ 40)N (Ct ≤ 40)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)Ct valuen/N (%)10018.65/5 (100)16.15/5 (100)17.385/5 (100)18.85/5 (100)195/5 (100)19.565/5 (100)20.818.419.1320.5719.7520.7823.421.122.0423.4723.2324.0524.722.223.5624.8524.1524.7822.62021.2822.7722.0122.9310121.55/5 (100)19.15/5 (100)21.015/5 (100)22.195/5 (100)22.45/5 (100)22.675/5 (100)23.821.222.5723.8723.6424.1326.624.425.4126.7126.7627.122.825.427.1228.1127.7528.2325.32324.8526.125.3425.8410224.95/5 (100)22.65/5 (100)24.335/5 (100)25.45/5 (100)25.345/5 (100)25.845/5 Open table in a new tab on time for Xpert Xpress was 1 the total turnaround time for Xpert Xpress was 51 minutes. However, only samples can be loaded onto the machinery at one on time for the BioGerm kit was 1 including RNA extraction and of mixture. There be 1 30 minutes for the real-time RT-PCR on on time for the Sansure kit was 2 and the real-time RT-PCR time was 1 30 minutes on the the BioGerm kit and the Sansure kit can test specimens at one Because SARS-CoV-2 is a that can from to To H. Yang J. F. Liu J. Cheng H. A of pneumonia with the 2019 novel coronavirus a study of a 2020; 395: Full Text Full Text PDF PubMed Scopus Google Scholar testing methods are for outbreak and There were to including clinical computed tomography, antibodies testing, and nucleic acid detection from clinical However, patients with COVID-19 cases are have only mild have on computed tomography, and have negative antibodies at the It is to a of COVID-19 positive nucleic acid detection is the only and the standard to The routine for nucleic acid is Until now, of real-time RT-PCR kits have been approved for use in testing by the NMPA. However, the routine real-time RT-PCR for diagnosis of is to in clinical is on the clinical and the who have been and by specific It several after nucleic acid purification for a reaction on to result in this assay the of patients testing, which is to by such as R. M.J. E. R. for and PubMed Scopus Google Scholar that are performed at or a patient and at the care or is In the United several sample-to-answer molecular platforms have been emergency use authorization by the to detect W. Smith E. Manji R. Schron D. Berry G.J. Clinical evaluation of three sample-to-answer platforms for the detection of SARS-CoV-2.J Clin Microbiol. 2020; 58: e00783-20Crossref PubMed Scopus (182) Google Scholar In China, the Nucleic Acid Detection and to detect SARS-CoV-2 Shanghai, China) has been by the Chinese The of this study was to the performance between sample-to-answer platform and routine real-time RT-PCR Because of the good performance of Xpert W. Smith E. Manji R. Schron D. Berry G.J. Clinical evaluation of three sample-to-answer platforms for the detection of SARS-CoV-2.J Clin Microbiol. 2020; 58: e00783-20Crossref PubMed Scopus (182) Google Scholar the performance of this test was compared with two routine real-time RT-PCR kits (BioGerm kit and Sansure kit) in this study. The clinical sensitivity was 100% for Xpert Xpress, 96.15% for the BioGerm kit, and 90% for the Sansure kit (Table 1). By the of two the Ct values of N2 were to be and for Xpert Xpress, both of which were considered as the Ct values of N were and for the Sansure kit, and were to be negative according to the (Table 2). In the detection of serial dilution samples from five positive specimens, when the dilution ratio was the Xpert Xpress had better detection capability. The were according to the of the kits 3 and In addition, it was to the Xpert Xpress platform for SARS-CoV-2 detection. the use of a transfer 300 μL of specimen was transferred from the viral transport medium tube into the in the then the cartridge lid was closed and the cartridge loaded onto the machinery. The test was then 50 minutes the was from the were a nucleic acid and and results analysis, a of individuals can the including laboratory emergency or However, Xpert Xpress is a detection of a single and only one is in laboratory. The BioGerm kit and Sansure kit are for detection, and the throughput of each is samples. For of a routine real-time RT-PCR assays can be and for emergency cases at the point of a sample-to-answer molecular platform be the better of the LOD or more sensitivity than the BioGerm kit and Sansure kit and of the target Xpert Xpress can be used to the However, this study has some this study not have positive only 26 positive specimens were evaluate the of the The LOD of the real-time RT-PCR kits on the of the nucleic acid purification kit. If the version of the nucleic acid purification kits (catalog number is used to the LOD of the Sansure kit can be as as 100 copies/mL not are of routine real-time RT-PCR by the for SARS-CoV-2 detection, with only two of used in this study. In compared the analytical and clinical performance and the workflow of the FDA-cleared Xpert Xpress SARS-CoV-2 assay platform with two NMPA-cleared routine real-time RT-PCR The Xpert Xpress SARS-CoV-2 assay platform was to have better clinical performance and sensitivity compared with routine real-time In addition, the Xpert Xpress SARS-CoV-2 assay platform is more to but is by Xpert Xpress, a sample-to-answer molecular platform for detection, can be to detect emergency cases. real-time RT-PCR with detection are for of This study provides for diagnosis methods. Cepheid for the an study agreement and Sansure for the nucleic acid purification and detection

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