key: cord-295957-s17z2ccf
authors: Bordi, Licia; Piralla, Antonio; Lalle, Eleonora; Giardina, Federica; Colavita, Francesca; Tallarita, Monica; Sberna, Giuseppe; Novazzi, Federica; Meschi, Silvia; Castilletti, Concetta; Brisci, Angela; Minnucci, Giulia; Tettamanzi, Veronica; Baldanti, Fausto; Capobianchi, Maria Rosaria
title: Rapid and sensitive detection of SARS-CoV-2 RNA using the Simplexa™ COVID-19 direct assay
date: 2020-05-04
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104416
sha: 
doc_id: 295957
cord_uid: s17z2ccf

BACKGROUND: So far, one of the major drawbacks of the available molecular assays for the diagnosis of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) is the need for viral nucleic acid extraction from clinical specimens. OBJECTIVE: The aim of this study was to evaluate the performances of a newly designed real-time RT-PCR (Simplexa™ COVID-19 Direct assay), that is established with an all-in-one reagent mix and no separate extraction required. RESULTS: The lower limit of detection (LOD) for both target genes resulted the same: 3.2 (CI: 2.9–3.8) log10 cp/mL and 0.40 (CI: 0.2–1.5) TCID50/mL for S gene while 3.2 log10 (CI: 2.9–3.7) log10 cp/mL and 0.4 (CI: 0.2–1.3) TCID50/mL for ORF1ab. The LOD obtained with extracted viral RNA for both S gene or ORF1ab was 2.7 log10 cp/mL. Crossreactive analysis performed in 20 nasopharyngeal swabs confirmed a 100% of clinical specificity of the assay. Clinical performances of Simplexa™ COVID-19 Direct assay were assessed in 278 nasopharyngeal swabs tested in parallel with Corman's method. Concordance analysis showed an "almost perfect" agreement in SARS-CoV-2 RNA detection between the two assays, being κ = 0.938; SE = 0.021; 95% CI = 0.896-0.980. CONCLUSIONS: The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, enabling highspeed detection in just over one hour, which is significantly faster than the up to five hours currently required by traditional extraction followed by amplification technologies, thus allowing prompt decision making regarding isolation of infected patients.

The pandemic caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) has generated global concern given its rapid spread in multiple countries and fatal progression of the Coronavirus infection disease (COVID-19) (1, 2) . However, asymptomatic or paucisymptomatic cases especially in the early phase of the infection have also been reported (3, 4) . Since the spread of SARS-CoV-2 out of China, one of the main concerns on the diagnosis of this new virus has been the development of rapid and sensitive diagnostic assays. So far, nucleic acid amplification testing is the gold standard for the diagnosis of SARS-CoV-2 in respiratory samples (5, 6) . Molecular assays as previously demonstrated for epidemics caused by other coronaviruses (SARS-CoV and MERS-CoV), are crucial for rapid application of infection control measures, case identification and contact tracing (6, 7) . However, one of the major drawbacks of these assays is the need for viral nucleic acid extraction from clinical specimens. Recently, DiaSorin S.p.A. (Gerenzano, Italy) has developed a SARS-CoV-2specific real-time RT-PCR (Simplexa™ COVID-19 Direct assay) performed directly on clinical samples.

This study was performed at two regional reference laboratories, with the following aims: I) to assess the analytical performances of the newly designed Simplexa™ COVID-19 Direct assay; II) to evaluate its clinical performance as compared to WHO protocols (7, 8) using different types of clinical specimens from patients with laboratory-confirmed COVID-19.

Viral stock used to establish analytical sensitivity was the first Italian isolate obtained from a patient hospitalized in the Biosafety Level 3 facility at National Institute for Infectious Diseases "Lazzaro Spallanzani" (INMI) in Rome, named 2019-nCoV/Italy-INMI1 (9) . The infectious titre of the viral stock used in the study, performed by Reed and Muench method on VeroE6 cells, was 10 7 TCDI 50 /mL; for the establishment of analytical sensitivity, the viral stock was spiked into oral swab-UTM matrix and serial dilutions (from 100 to 0.001 TCDI 50 /mL) were tested in multiple replicates. The evaluation of the corresponding RNA copies/mL was performed based on standard curve obtained by Corman"s E-SARS-CoV-2 gene.

A total of 278 consecutive respiratory samples (nasal and nasopharyngeal swabs) collected for COVID-19 diagnosis between 20 February and 24 March 2020 were included in the study; twenty additional nasopharyngeal swabs from patients known to be positive for other Human Coronaviruses were used to establish clinical specificity of the Simplexa™ COVID-19 Direct assay. Moreover, to evaluate the performance of the test in a different clinical specimen, a total of 33 Broncho-Alveolar Lavage (BAL) collected for COVID-19 diagnosis between 20 March and 03 April 2020 were also analysed in parallel with the Simplexa™ COVID-19 Direct assay and the routine laboratory method, based on the WHO protocols (7, 8) , using the Abbot m2000 extraction platform.

J o u r n a l P r e -p r o o f For Simplexa™ COVID-19 Direct assay, one vial of Reaction Mix was thawed for each sample.

Respiratory samples were collected with a flexible nasopharyngeal nylon flocked swabs (FLOQSwabs™, Copan Italia, Brescia, Italy) dipped in 2-3 mL universal transport medium (UTM™, Copan Italia). After swirling, the swab was discarded and 50μL of sample and 50μL of Reaction Mix were added to their specific wells on a direct amplification disk, which was loaded onto the LIAISON® MDX instrument (DiaSorin). For BAL, a pre-treatement with 1:1 SV40 Sputagest Selectavial (Mast Diagnostic GmbH, Reinfeld, Germany) was performed.

Total nucleic acids (DNA/RNA) were extracted from 200μL of nasal and nasopharyngeal specimens.

Clinical samples were pre-treated with 1:1 ATL lysis buffer and extracted using the QIAsymphony® instrument with QIAsymphony® DSP Virus/Pathogen Midi Kit (Complex 400 protocol) according to the manufacturer"s instructions (QIAGEN, Qiagen, Hilden, Germany). The presence of SARS-CoV-2 RNA was assessed by Corman"s protocol, targeting E and RNA-dependent RNA polymerase (RdRp) genes as reference method (7) . According to the WHO guidelines Corman"s protocol was carried out using the E assay for screening and the RdRp assay for confirmation (8). Total nucleic acids (DNA/RNA) were extracted from 600μL of BAL samples, pre-treated with 1:1 SV40 Sputagest Selectavial and then with 1:1 ATL lysis buffer, using the Abbott m2000 system according to the manufacturer"s instructions (Abbott molecular, Des Plaines, IL, USA).

The analytical sensitivity (SARS-Cov-2 copy number at a 95% detection rate and SARS-Cov-2 TCDI 50 at a 95% detection rate) was calculated with probit analysis, using the MedCalc statistical software, on 

To assess the sensitivity of the prototype assay, both live SARS-CoV-2 and extracted viral RNA were tested. More in detail, for LOD calculation with live virus, SARS-CoV-2 particles from 2019nCoV/Italy-INMI1 isolate were spiked into oral swab-UTM matrix and serial dilutions from 100 to 0.001 TCDI 50 /mL were tested in replicates. We observed 100% detection down to about 1 TCID50/ml, corresponding to 4x10 3 cp/mL ( Table 1 ). The analytical sensitivity (i.e. the LOD, corresponding to the concentration of SARS-CoV-2-RNA detected with response probability of 95% for either S or ORF1ab) was determined by Probit regression model (Fig. 1 ). More in detail, the LOD for both target genes resulted the same: 3.2 (CI: 2.9 to 3.8) log 10 cp/mL (Fig. 1a) TCID 50 /mL (Fig. 1d) for ORF1ab. Consistent, although slightly better results, were obtained for LOD calculated on extracted viral RNA. This was spiked in universal transport medium (UTM) additioned with RNasin (1.6 U/µL) to avoid RNA degradation, and replicate 10-fold serial dilutions of were loaded on the MDx instrument and tested with the Simplexa™COVID-19 Direct assay. The LOD obtained with extracted viral RNA for both S gene or ORF1ab was 2.7 log 10 cp/mL.

In silico evaluation and preliminary in vitro analytical specificity study showed that the (Table 3) . Moreover, the performance of the assay in a different clinical specimen type, i.e. BAL, that is considered of utmost importance in patients with lower respiratory tract implication, was also evaluated: a total of 33 BAL samples were analysed in parallel with the Simplexa™ COVID-19 Direct assay and Corman"s method. For these samples, the Abbot m2000 extraction platform was used, due to temporary restriction of QIAsymphony® reagents (Table   4) 

Since Furthermore, the simplicity of design and the all-in-one coupled with easy-to-use design, makes it suitable for the field settings, and for near-to-patient diagnosis.

The LOD of the prototype assay, established with both SARS-CoV-2 viral particles and SARS-CoV2 extracted viral RNA, was less than 1,000 cp/ml. All the SARS-CoV-2 samples resulted positive with the reference test (Corman"s method), also resulted positive with Simplexa™ COVID-19 Direct assay, with a diagnostic sensitivity of 100% and "almost perfect" concordance. Notably, Simplexa™ COVID-19 Direct assay showed a slightly higher sensitivity with respect of the reference test, identifying near 3% additional positive samples. An high performance of the test was also observed using different clinical specimens: among the BAL samples that were able to be analysed by both methods, a perfect agreement was observed; noteworthy, Simplexa™ COVID-19 Direct assay was able to detect the virus in BAL samples resulted invalid with the reference method. The discrepancy in the processing capability between the two systems is attributable to the highly viscous composition of BAL, that could impair its accessibility to a traditional extraction method compared to a faster and easier sample processing method such as that used in the Simplexa™ COVID-19 Direct assay. Moreover a 100% of clinical specificity was observed against swabs resulted positive to other viruses, particularly human coronaviruses, indicating no cross-reactivity with other similar viruses. Key advantages of this assay are simple operation procedures and high-speed of detection in just over an hour, which is significantly faster than the up to seven hours currently required by traditional extraction followed by amplification technologies. The lack of extraction reduces turnaround time (TAT) for the diagnosis of COVID -19, thus allowing prompt decision making regarding isolation of infected patients.

The only limitation of the assay is the small number of samples which can be tested in a run, since each instrument can support a ring of maximum eight position; this limitation is offset by the rapidity of the J o u r n a l P r e -p r o o f assay, due to the lack of extraction. Overall, the good analytical and clinical performances, coupled with user friendly architecture and short TAT indicate that it is promising for laboratory diagnosis of COVID-19 and field application.

Ethical approval was not required as human samples were routinely collected and patients' data remained anonymous. J o u r n a l P r e -p r o o f 

Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia

A Novel Coronavirus from Patients with Pneumonia in China

Novel Coronavirus Infection in Hospitalized Infants Under 1 Year of Age in China

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster

Coronavirus as a possible cause of severe acute respiratory syndrome

Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease

Detection of 2019 novel coronavirus (2019-nCoV) by realtime RT-PCR

Molecular characterization of SARS-CoV-2 from the first case of COVID-19 in Italy

This research was supported by funds to National Institute for Infectious Diseases "Lazzaro Spallanzani" IRCCS from Ministero della Salute, Ricerca Corrente, linea1; European Commission -Horizon 2020 (EU project 101003544 -CoNVat; EU project 101003551 -EXSCALATE4CoV) and to
