Production and Performance Assessment of a Severe Acute Respiratory Syndrome Coronavirus 2 Biomimetic in a Verification Program for Pandemic Readiness

During the early stages of the 2019 coronavirus disease (COVID-19) pandemic in South Africa, one of many challenges included availability of control material for laboratory proficiency testing programs. Proficiency testing control material using live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or RNA extracted from cell culture was either biohazardous or costly, particularly in resource-limited settings. This study reports the development and application of a noninfectious SARS-CoV-2 biomimetic Mycobacterium smegmatis strain that mimics a positive result in the GeneXpert SARS-CoV-2 Xpert Xpress cartridge. Nucleotide sequences located in genes encoding the RNA-dependent RNA polymerase, the nucleocapsid, and the envelope proteins were used. The resulting biomimetic strain was prepared as a positive proficiency testing control and distributed in South Africa for verification of laboratories before their testing of clinical specimens. Between April and December 2020, a total of 151 GeneXpert instruments with 2532 modules were verified to bring COVID-19 mass testing in South Africa online. An average concordance of 98.6% was noted in the entire laboratory network, allowing identification of false-positive/false-negative results and instrument errors. This noninfectious, easily scalable proficiency testing control material became available within 2 months after the start of the pandemic in South Africa and represents a useful approach to consider for other diseases and future pandemics. During the early stages of the 2019 coronavirus disease (COVID-19) pandemic in South Africa, one of many challenges included availability of control material for laboratory proficiency testing programs. Proficiency testing control material using live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or RNA extracted from cell culture was either biohazardous or costly, particularly in resource-limited settings. This study reports the development and application of a noninfectious SARS-CoV-2 biomimetic Mycobacterium smegmatis strain that mimics a positive result in the GeneXpert SARS-CoV-2 Xpert Xpress cartridge. Nucleotide sequences located in genes encoding the RNA-dependent RNA polymerase, the nucleocapsid, and the envelope proteins were used. The resulting biomimetic strain was prepared as a positive proficiency testing control and distributed in South Africa for verification of laboratories before their testing of clinical specimens. Between April and December 2020, a total of 151 GeneXpert instruments with 2532 modules were verified to bring COVID-19 mass testing in South Africa online. An average concordance of 98.6% was noted in the entire laboratory network, allowing identification of false-positive/false-negative results and instrument errors. This noninfectious, easily scalable proficiency testing control material became available within 2 months after the start of the pandemic in South Africa and represents a useful approach to consider for other diseases and future pandemics. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of 2019 coronavirus disease (COVID-19), was first described in December 2019, subsequently progressing to a global pandemic (Figure 1). It was initially designated as 2019 novel coronavirus but subsequently renamed.1Coronaviridae Study Group of the International Committee on Taxonomy of VirusesThe species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.Nat Microbiol. 2020; 5: 536-544Crossref PubMed Scopus (4798) Google Scholar, 2WHOSurveillance Case Definitions for Human Infection with Novel Coronavirus (nCoV): Interim Guidance, 11 January 2020. World Health Organization, Geneva, Switzerland2020Google Scholar, 3Ren L.L. Wang Y.M. Wu Z.Q. Xiang Z.C. Guo L. Xu T. et al.Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study.Chin Med J (Engl). 2020; 133: 1015-1024Crossref PubMed Scopus (831) Google Scholar, 4Wu F. Zhao S. Yu B. Chen Y.M. Wang W. Song Z.G. Hu Y. Tao Z.W. Tian J.H. Pei Y.Y. Yuan M.L. Zhang Y.L. Dai F.H. Liu Y. Wang Q.M. Zheng J.J. Xu L. Holmes E.C. Zhang Y.Z. A new coronavirus associated with human respiratory disease in China.Nature. 2020; 579: 265-269Crossref PubMed Scopus (7237) Google Scholar, 5Wu F. Zhao S. Yu B. Chen Y.M. Wang W. Song Z.G. Hu Y. Tao Z.W. Tian J.H. Pei Y.Y. Yuan M.L. Zhang Y.L. Dai F.H. Liu Y. Wang Q.M. Zheng J.J. Xu L. Homes E.C. Zhang Y.Z. Author correction: a new coronavirus associated with human respiratory disease in China.Nature. 2020; 580: E7Crossref PubMed Scopus (143) Google Scholar, 6Zhu 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. China Novel Coronavirus Investigating and Research TeamA novel coronavirus from patients with pneumonia in China, 2019.N Engl J Med. 2020; 382: 727-733Crossref PubMed Scopus (17880) Google Scholar It is a ß-coronavirus with a single-stranded RNA genome of 29,903 nucleotides (Strain Wuhan-Hu-1; GenBank accession number MN908947.3, https://www.ncbi.nlm.nih.gov/nuccore). Early during the pandemic, it became evident that effective control required fast and accurate diagnosis, as the inability to rapidly identify infected/diseased individuals fueled further transmission. To detect the SARS-CoV-2 genome in clinical specimens requires nucleic acid amplification technologies. Sequence information and assay methods for early diagnostic assays were made publicly available to assist global health systems with the implementation of pandemic control measures.7World Health OrganizationMolecular assays to diagnose COVID-19. WHO, Geneva, Switzerland2020https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdfDate accessed: October 21, 2023Google Scholar Global deployment of nucleic acid amplification technologies, particularly in resource-limited settings, was difficult at the time because of constraints on tools, materials, trained technicians, and laboratory resources.8Reusken C.B.E.M. Broberg E.K. Haagmans B. Meijer A. Corman V.M. Papa A. Charrel R. Drosten C. Koopmans M. Leitmeyer K. EVD-LabNet and ERLI-NetLaboratory readiness and response for novel coronavirus (2019-nCoV) in expert laboratories in 30 EU/EEA countries, January 2020.Euro Surveill. 2020; 252000082Crossref Scopus (128) Google Scholar The lack of robust, consistent proficiency testing (PT) controls to implement and verify the performance of these emerging SARS-CoV-2 diagnostics was a significant barrier to reliable mass testing. At the time, PT control materials were either clinical specimens with a confirmed positive result or purified viral RNA from cell culture. This was problematic and costly because of inherent biohazard concerns, instability of RNA, the need for cold chain storage, and/or limited availability. We previously reported the use of modified, noninfectious derivative strains of Mycobacterium smegmatis for use in instrument verification and external quality assurance in tuberculosis nucleic acid amplification technology diagnostics programs, which faced similar challenges with respect to biologically safe PT controls.9Machowski E.E. Kana B.D. Genetic mimetics of Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus as verification standards for molecular diagnostics.J Clin Microbiol. 2017; 55: 3384-3394Crossref PubMed Scopus (3) Google Scholar These biomimetic strains were able to mimic positive tuberculosis diagnosis on two distinct platforms: GeneXpert technology (Cepheid, Sunnyvale, CA) and the Hain Lifescience line probe assay (Hain Lifescience, Nehren, Germany). In addition, this approach also demonstrated utility for detection of a Staphylococcus aureus target sequence on the GeneXpert platform. On the basis of the sequence of genome targets available during 2020 at the time of the COVID-19 outbreak, this biomimetic approach was adapted to generate noninfectious PT control material that was easily scalable in South Africa. It was used by SmartSpot Quality (Pty) Ltd. (Johannesburg, South Africa) to generate PT controls in its verification procedure, which was implemented in 2020 to verify the competency of laboratories to test for SARS-CoV-2. This approach can easily be adapted for a variety of established or emerging diseases, and it holds promise for future pandemics because of its rapid design and production capabilities. All strains and plasmids used and generated in this study are listed in Table 1.10Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.Mol Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (1038) Google Scholar,11Rauzier J. Moniz-Pereira J. Gicquel-Sanzey B. Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum.Gene. 1988; 71: 315-321Crossref PubMed Scopus (81) Google Scholar Strain Escherichia coli DH5α was grown at 37°C shaking in standard lysogeny broth or on Luria agar, supplemented with 50 μg/mL of kanamycin where applicable. M. smegmatis strains were grown at 37°C shaking in Difco Middlebrook 7H9 liquid medium (Becton, Dickinson and Company, Sparks, MD) supplemented with 0.085% NaCl, 0.2% glucose, 0.2% glycerol, and 0.05% Tween-80 or on Difco Middlebrook 7H10 solid medium (Becton, Dickinson and Company) supplemented with 0.085% NaCl, 0.2% glucose, and 0.5% glycerol. Kanamycin was used at 25 μg/mL.Table 1Plasmids and Bacterial Strains Generated or Used in this StudyPlasmid nameDescriptionReferencepRRNE∗Targets for RdRP1, RdRP2, the N protein (N1-N3-N2), and E protein were all included on the plasmid; however, the GeneXpert SARS-CoV-2 Xpert Xpress cartridge tests only for N2 and E.Commercial plasmid bearing targets for RdRP1, RdRP2, N protein, and E protein of SARS-CoV-2, aphThermo Fisher Scientific, GeneArt GmbH (Regensburg, Germany)pYUB12Escherichia coli and mycobacterial shuttle vector, oriM from Mycobacterium fortuitum plasmid pAL5000, aph10Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.Mol Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (1038) Google Scholar,11Rauzier J. Moniz-Pereira J. Gicquel-Sanzey B. Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum.Gene. 1988; 71: 315-321Crossref PubMed Scopus (81) Google ScholarpRRNE-oripRRNE with the mycobacterial origin of replication of plasmid pAL5000; targets for RdRP1, RdRP2, N protein, and E protein of SARS-CoV-2, aphThis workStrainsDescriptionReferenceEscherichia coli DH5-αfhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17Promega (Madison, WI)Mycobacterium smegmatis mc2155ept-1; High-frequency transformation mutant of M. smegmatis ATCC (Manassas, VA) 60710Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.Mol Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (1038) Google Scholar mc2155-rrneDerivative of mc2155 bearing shuttle plasmid pRRNE-ori; targets for RdRP1, RdRP2, N protein, and E protein of SARS-CoV-2, KanThis work mc2155-SARSnegDerivative of mc2155 bearing an integrating plasmid with no SARS-CoV-2 targets, KanThis workE, envelope protein; Kan, kanamycin; N, nucleoprotein; RdRP, RNA-dependent RNA polymerase; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.∗ Targets for RdRP1, RdRP2, the N protein (N1-N3-N2), and E protein were all included on the plasmid; however, the GeneXpert SARS-CoV-2 Xpert Xpress cartridge tests only for N2 and E. Open table in a new tab E, envelope protein; Kan, kanamycin; N, nucleoprotein; RdRP, RNA-dependent RNA polymerase; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. The complete genomic organization of SARS-CoV-2 is shown in Figure 2A, and the selected target regions for generating the biomimetic strain are shown in Figure 2B. Plasmid pRRNE was purchased commercially and carried the target sequences for the regions in the SARS-CoV-2, internal to the genes encoding the RNA-dependent RNA polymerase (RdRP1 and RdRP2), the nucleoprotein (N1-N3-N2), and the envelope protein (E). These sequences were made publicly available in January 2020.12Corman V.M. Landt O. Kaiser M. Molenkamp R. Meijer A. Chu D.K. Bleicker T. Brünink S. Schneider J. Schmidt M.L. Mulders D.G. Haagmans B.L. van der Veer B. van den Brink S. Wijsman L. Goderski G. Romette J.L. Ellis J. Zambon M. Peiris M. Goossens H. Reusken C. Koopmans M.P. Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.Euro Surveill. 2020; 25: e00599-e00620Crossref Scopus (4842) Google Scholar pRRNE-ori was introduced into M. smegmatis, in combination with a mycobacterial origin of replication by standard electroporation method,10Snapper S.B. Melton R.E. Mustafa S. Kieser T. Jacobs Jr., W.R. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.Mol Microbiol. 1990; 4: 1911-1919Crossref PubMed Scopus (1038) Google Scholar to yield strain mc2155-rrne as a positive PT control. M. smegmatis strain mc2155-SARSneg, lacking the target sequences for the SARS-CoV-2 cartridge, was used as the PT negative control. Before distribution, PT controls were validated at a central laboratory. They were tested on 2 independent days by two separate individuals to ensure consistency of the material. The amounts of biomimetic material included in a positive PT control were adjusted to yield cycle threshold (CT) values of 35 ± 2 for target E and 38 ± 2 for target N2. Verification panels contained two PT controls each, one positive and one negative (Figure 3). One verification panel was distributed to each GeneXpert instrument in laboratories enrolled in the SmartSpot Quality SARS-CoV-2 verification program. Each affiliated laboratory exported the test results from its GeneXpert instrument via a cloud-based interface to SmartSpot Quality (Pty) Ltd. (Figure 3). The results were auto-analyzed on SSQmonitor, and verification reports were released in real time. Where results were not concordant, reports were held back for review before report finalization. Where warranted, CT values were analyzed to facilitate accurate result scoring and to identify and address potential root causes for incorrect results. PT controls were validated in the SARS-CoV-2 Xpert Xpress cartridge (GeneXpert Dx software version 4.7b) before launching the control in testing laboratories. They yielded the expected result of positive (Figure 2C), based on the presence of the E and N2 targets at the correct range of CT values 35 ± 2 for target E, and 38 ± 2 for target N2. PT negative controls with CT values of 0.0 for both E and N2 yielded the expected result of negative (Figure 2D). Verification panels were shipped to laboratories to bring GeneXpert modules online for testing, where one module is the unit in a GeneXpert machine that can test one cartridge at a time (Figure 3). The large demand for SARS-CoV-2 testing and reagents, together with lockdown regulations, limited access to resources, which were accordingly prioritized for patient testing. To minimize the number of cartridges required to verify a laboratory for SARS-CoV-2 testing, machines that were already enrolled for PT programs relating to other diseases, such as tuberculosis, received only one positive and one negative PT control for SARS-CoV-2 verification. Sixty-five laboratories across South Africa were enrolled in the verification program with a total of 151 instruments, which effectively comprised 2532 modules. A total of 702 controls were distributed, from which laboratories tested 349 positive PT controls and 350 negative PT controls. The data from these controls are reported in Table 2. Discrepancies in the numbers of PT controls submitted indicate that some laboratories did not test both PT controls (positive and negative) supplied within the verification panel. This was attributed to reagent shortages, human error in processing the PT control/s, and/or human error in submitting both results.Table 2Test Result of Xpert Xpress SARS-CoV-2 AnalysisVariablePT controls tested, NCorrect, n (%)Incorrect, n (%)PositivePresumptive∗A presumptive positive result indicates a test that only flagged positive for the e gene, which was indicative of any β-coronavirus, not only SARS-CoV-2.NegativeInstrument errorPositive PT control349346 (99.1)NA0 (0.0)3 (0.9)1 (0.3)Negative PT control350343 (98.0)6 (1.7)1 (0.3)NA0 (0.0)NA, not applicable; PT, proficiency testing; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.∗ A presumptive positive result indicates a test that only flagged positive for the e gene, which was indicative of any β-coronavirus, not only SARS-CoV-2. Open table in a new tab NA, not applicable; PT, proficiency testing; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. The verification panels yielded 99.1% (346/349) correct positive results and 98.0% (343/350) correct negative results, with an average concordance of 98.6% (Table 2). The incorrect results submitted for the negative PT controls were as follows: 1.7% (6/350) reported as positive and 0.3% (1/350) reported as presumptive positive (a CT value was obtained for analyte E and no CT value was obtained for N2). The incorrect results submitted for the positive PT controls were as follows: 0.9% (3/349) reported as negative and 0.3% (1/349) reported as an instrument error. To assess the proficiency of testing facilities, a GeneXpert system was deemed fit for purpose provided that all modules that were tested with the panel passed verification. If any module did not pass verification, a repeated PT control was tested. In this work, the utility of using a noninfectious biomimetic to generate biologically safe COVID-19 diagnostic PT controls, and integrating these into verification of a molecular diagnostic assay, was demonstrated. The resulting biomimetics were robust and easily scalable as PT control material for laboratories across an entire country. This approach enabled activation of 2532 GeneXpert modules for immediate SARS-CoV-2 mass testing. The application of this technology for SARS-CoV-2 can be applied to other diagnostic platforms, based on nucleic acid amplification technology approaches. However, conditions that ensure lysis of the bacterial biomimetic cell wall may require some optimization on other platforms. Given the robust results obtained in this study, this issue did not emerge in the GeneXpert platform. In the field, SARS-CoV-2 Xpert Xpress performed well in side-by-side comparisons with other diagnostic platforms to detect the virus on clinical samples.13Loeffelholz M.J. Alland D. Butler-Wu S.M. Pandey U. Perno C.F. Nava A. Carroll K.C. Mostafa H. Davies E. McEwan A. Rakeman J.L. Fowler R.C. Pawlotsky J.M. Fourati S. Banik S. Banada P.P. Swaminathan S. Chakravorty S. Kwiatkowski R.W. Chu V.C. Kop J. Gaur R. Sin M.L.Y. Nguyen D. Singh S. Zhang N. Persing D.H. Multicenter evaluation of the cepheid Xpert xpress SARS-CoV-2 test.J Clin Microbiol. 2020; 58: e00926-e01020Crossref PubMed Scopus (126) Google Scholar,14Cao X.J. Fang K.Y. Li Y.P. Zhou J. Guo X.G. The diagnostic accuracy of Xpert xpress to SARS-CoV-2: a systematic review.J Virol Methods. 2022; 301114460Crossref Scopus (7) Google Scholar This is likely because of the high reliability of the SARS-CoV-2 Xpert Xpress assay, where the reverse transcription and PCR steps are integrated and robust within a single cartridge. In this context, the biomimetic PT controls reported herein are DNA based and, as such, do not control for efficiency of the reverse transcription step. A critical factor in the ease of implementation in the rollout of diagnostic capability in South Africa was that GeneXpert technology already had a large presence because of its extensive use for tuberculosis diagnosis.15Scott L.E. McCarthy K. Gous N. Nduna M. Van Rie A. Sanne I. Venter W.F. Duse A. Stevens W. Comparison of Xpert MTB/RIF with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high HIV prevalence setting: a prospective study.PLoS Med. 2011; 8e1001061Crossref Scopus (142) Google Scholar For instrument verification, it was shown that the results obtained from the biomimetic M. smegmatis SARS-CoV-2 PT control material reliably mimic those expected from corresponding clinical specimens, based on the presence/absence of the targets included in the material. This approach can be easily adapted for emerging pathogens and future pandemics and negates the requirement to handle large-scale infectious specimens in PT programs. Given the increased regulation on growing pathogens in laboratories, this approach holds promise for bolstering expanded rollout of molecular diagnostics. E.E.M. and B.D.K. conceived the study and designed the biomimetic strain; E.E.M. performed laboratory to generate mc2155-rrne and and and proficiency testing control panels and the the data and the data of the All the B.D.K. and E.E.M. a on this work on which are no number and a from SmartSpot Quality (Pty) and are by SmartSpot Quality (Pty)