key: cord-268993-2sjh17mw
authors: Rödel, Jürgen; Egerer, Renate; Suleyman, Aynur; Sommer-Schmid, Beatrice; Baier, Michael; Henke, Andreas; Edel, Birgit; Löffler, Bettina
title: Use of the variplex(TM) SARS-CoV-2 RT-LAMP as a rapid molecular assay to complement RT-PCR for COVID-19 diagnosis
date: 2020-08-31
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104616
sha: 
doc_id: 268993
cord_uid: 2sjh17mw

BACKGROUND: Molecular assays based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) may be useful for rapid diagnosis of the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) because of the easy performance and the option to bypass RNA extraction. OBJECTIVES: This study was designed to evaluate the clinical performance of the CE-labeled variplexTM real time SARS-CoV-2 RT-LAMP assay in comparison to commercial RT-PCRs. STUDY DESIGN: RNA extracted from pharyngeal swabs was tested by variplex™ RT-LAMP and Corman’s LightMix™ E gene RT-PCR as reference. Samples of respiratory secretions from Coronavirus infection disease (COVID-19) and negative control patients were analyzed by variplex™ without RNA extraction and tested in parallel with the Allplex™ and VIASURE BD MAX RT-PCRs. RESULTS: Using isolated RNA variplex™ RT-LAMP showed a sensitivity of 75% compared to LightMix E gene RT-PCR but contrary to the latter it produced no false-positive results. For the evaluation of samples from respiratory secretions concordance analysis showed only a moderate agreement between the variplex™ RT-LAMP conducted on unprocessed samples and Allplex™ and VIASURE RT-PCRs (Cohen’s κ ranging from 0.52-0.56). Using the approach to define a sample as true-positive when at least two assays gave a positive result the clinical sensitivities were as follows: 76.3% for variplex™, 84.2% for Allplex™ and 68.4% for VIASURE. However, when results of RT-PCR and RT-LAMP were combined diagnostic sensitivity was increased to 92-100%. CONCLUSION: The variplex RT-LAMP may serve as a rapid test to be combined with a RT-PCR assay to increase the diagnostic accuracy in patients with suspected COVID-19 infection.

The severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) pandemic has already caused an enormous burden on healthcare systems worldwide [ high analytical sensitivity several studies reported on false-negative as well as fluctuating results in patients whose clinical diagnosis using chest CT was in accordance with COVID-19 [2, 7] . Problems with clinical sensitivity of nucleic acid amplification tests can be due to analytical errors of RNA isolation procedures and choose of inadequate primers. Other challenges in diagnostics are associated with the significantly increased requests for testing, resulting in time delays to generate diagnostic reports [8, 9] . Moreover, mass testing has rapidly caused serious shortages in the supply of RNA purification kits in many countries [9, 10] . For a rapid diagnosis of SARS-CoV-2 cost-effective methods with low hands-on time that circumvent limitations of RT-PCR may be helpful tools for a routine diagnostic workflow [9, 11] . RT-loop-mediated isothermal amplification (LAMP) may offer the possibility to be established as an alternative diagnostic technique [12] [13] [14] . The combination of RT with Bst polymerase possessing a DNA strand displacement activity allows amplification of target genes at a constant temperature in less than one hour. RNA purification can be bypassed J o u r n a l P r e -p r o o f depending on the sample type and different transport media because of the robustness of the polymerase.

There are several studies that demonstrated satisfying sensitivity and specificity of RT-LAMP for SARS-CoV-2 detection but little is known about its performance of testing clinical samples directly without RNA extraction [11] [12] [13] [14] . In this study we evaluated the newly introduced CE-labeled variplex SARS-CoV-2 LAMP assay and compared the clinical performance with commercial RT-PCR tests. Testing was performed using pharyngeal washes and samples from respiratory secretions, including sputum, endotracheal secretions, and bronchoalveolar lavage.

Pharyngeal specimens were collected using eSwab™ transport systems (Copan, Brescia, Italy). Total viral RNA was extracted from 200 l of the sample medium using the QIAsymphony DSP Virus/Pathogen Mini Kit (Qiagen, Hilden, Germany. Extraction was performed on the automated Qiasymphony SP instrument (Qiagen). Purified RNA was eluted in 60 l AVE buffer and divided into two parts for testing. To rule out cross-reactivity with human coronaviruses 229E and OC43 external quality assessment samples (INSTAND e.V., Düsseldorf, Germany) were processed in a similar manner. Reference RT-PCR was performed using the LightMix Modular SARS-CoV E-gene primers (TIB Molbiol, Berlin, Germany) and the LightCycler Multiplex RNA Virus Master (Roche, Penzberg, Germany) [15] . RT-PCR was run on a LightCycler 480 (Roche, Penzberg, Germany).

The variplex SARS-CoV-2 is a qualitative molecular assay using a mix of 6 oligonucleotide primers targeting a 282-bp sequence of the membrane protein (M) gene. For a single test 15 l of RT master mix and 8 l of eluted RNA were pipetted into two wells of a Genie test strip (Amplex Diagnostics). 2 l of the primer mixes for SARS-CoV-2 or the inhibition control were added to one each well. Tests were run at 65 o C for 40 min using a Genie II Mk2A device (Amplex Diagnostics). Amplification was measured by real-time fluorescence detection using a DNA intercalating dye. Data interpretation and calculations were automatically performed by the integrated eazyReport TM software (Amplex Diagnostics). For the VIASURE assay 200 l of the sample was used for RNA extraction. VIASURE rehydration buffer and gene reaction tubes containing a ready-to-use master mix were loaded onto BD MAX ExK TNA-3 reagent strips. Nucleic acid extraction and real time RT-PCR were performed on the automated BD MAX system (BD).

To assess the analytical sensitivity of the assays the SARS-CoV-2 isolate Jena/2020/5159 propagated and titrated on Vero-76 cells was used. 10-fold serial dilutions of a virus stock of 10 7 TCID50/ml in a pharyngeal wash were mixed with Copan SL solution and processed for the different assays as described above.

The qualitative performance of the assays was assessed by calculating the specificity, sensitivity, negative and positive prospective values, and accuracy. For reference a sample was defined as true-positive when at least two different tests gave a positive result.

Concordance of two diagnostic tests was examined by Cohen's  coefficient analysis.

Pearson coefficient analysis.

First, we analyzed a panel of pharyngeal swabs sent to the laboratory for routine SARS-CoV-aliquots were tested. Samples with divergent results between LightMix RT-PCR and RT-LAMP were verified by VIASURE and Allplex assays in order to identify false-positively tested specimen. 10 out of 96 RNA eluates that were LightMix E-positive could not be confirmed by a second test and were defined as false-positive. Their median Ct value was 36.6 (IQR 36.1-37.6). In contrast, no false-positive results were observed using the variplex RT-LAMP. However, the sensitivity of RT-LAMP was only 75% ( To verify the sensitivity of RT-LAMP extracted RNA from a log-dilution series of a virus stock was tested. The variplex assay achieved a reliable detection at 1 TCID50/ml, corresponding to 0.03 TCID50/reaction. In comparison LightMix RT-PCR showed 100% detection down to 0.1 TCID50/ml. This concentration was positive by RT-LAMP in 33% of the samples (Table 2 ).

Next, we investigated a panel of clinical samples that were tested by RT-LAMP without RNA extraction. Only samples from respiratory secretions and pharyngeal washes were used because in preliminary experiments we observed inhibitory effects by transport media of swabs on RT. A total of 43 specimens collected from 20 patients were included. From 6 patients 3 or more samples were obtained during the course of the disease. As controls we examined 30 samples from patients that were repeatedly tested negative by LightMix screening RT-PCR. Respiratory secretions from COVID-19 patients were often highly viscous and tough. To homogenize the specimens they were mixed with Copan SL solution.

This procedure was applied to all samples to standardize the methodology. Homogenized samples diluted in LPTV buffer were directly pipetted into the master mix for RT-LAMP. For comparative analysis two aliquots were subjected to RNA isolation and RT-PCR using the J o u r n a l P r e -p r o o f Allplex and VIASURE BD MAX assays. All tests did not produce false-positives results in the group of control patients. From the samples of COVID-19 patients heterogeneous results were obtained. As expected a high agreement of results was found for the three different targets of the Allplex assay (Table 3) . The results obtained with the variplex RT-LAMP only showed a moderate agreement to both the Allplex and VIASURE RT-PCR results (Table 3) . To calculate how the moderate Cohen's  concordance coefficients were related to different sensitivities of the assays we defined a sample as true-positive when at least two target genes of the virus were detected. When only one target gave a positive signal the sample was tested by the LightMix RT-PCR to verify the result. Using this approach, all assay had sensitivities <90% (Table 4 ). The Allplex RdRP assay offered the highest sensitivity of 84%, followed by E and N gene tests from the same kit. Combining the three targets of Allplex did not result in a higher positive rate of the samples. The sensitivity of the VIASURE assay was only 68.4% and that of the variplex RT-LAMP was in between, at 76.3% (Table 4 ). However, when results of the variplex RT-LAMP were combined with those of the VIASURE S or Allplex RdRP RT-PCR diagnostic sensitivity was increased to 92 and 100%, respectively (Table 4) Table 5 shows the course of testing an ICU patient over 30 days, illustrating the fluctuating results by different assays.

In comparison the different sensitivities of the assays were also examined using simulated samples. For these experiments we started at 300 TCID50/ml because of the dilution of the samples in LPTV buffer for direct RT-LAMP testing. As shown in Table 6 the Allplex RT-PCRs reached higher sensitivities than the other assays. The lower sensitivity of the variplex RT-LAMP was probably caused by the relatively high dilution of the sample in LPTV buffer because the limit of detection of 0.004 TCID50/reaction was satisfying in comparison to the Allplex RT-PCR.

Timely and accurate laboratory diagnosis of patients with the suspicion of SARS-CoV-2 infection is important for optimizing patient treatment and preventing transmission to other persons [3]. RT-PCR is the standard method to detect an acute infection and is also used to identify asymptomatic carriers [16, 17] . However, several studies have reported false-negative results in initial testing of symptomatic patients as well as during the course of the disease in no small measure that can have an impact on isolation or discharge of patients [2, 7]. It has been suggested that a single RT-PCR assay should not be the only laboratory diagnostic marker [7, 16] . The data of this study demonstrate that the variplex LAMP SARS-CoV-2 assay may be suitable as an additional tool to close gaps in COVID-19 diagnosis.

By using extracted RNA the variplex RT-LAMP assay showed a lower sensitivity, compared to our screening E gene RT-PCR, but performance was acceptable when only E gene Ct values <35 were considered. This cut-off has been chosen because on one hand it has been proposed that patients diagnosed with high Ct values are rather non-infectious and on the other hand we could identify several false-positive RT-PCR tests that were associated with a high Ct value [18] .

A major advantage of RT-LAMP is that it allows a simple testing of specimens when unprocessed samples are used, bypassing the bottleneck of RNA extraction [9, 19] . Against J o u r n a l P r e -p r o o f the background of irregularities regarding the delivery of RNA isolation kits by many manufacturers RT-LAMP would be highly attractive as an alternative easy-to-use technology [9, 13] . For direct testing we focused on samples from respiratory secretions and pharyngeal washes instead of swabs because several transport media can inhibit or reduce RT activity, as reported in recent studies [10, 11] . Another reason was that the supply of swabs with fluid transport media was running into a critical shortage in a phase of significantly increased demand for testing. . By using RT-LAMP to test unprocessed samples this problem is avoided but RT activity may be inhibited by carbohydrates and salts depending on the sample composition [10] . In this context suitable specimen types have to be carefully evaluated. Saliva which has been described to contain high virus copy numbers may also represent a potential specimen type for direct RT-LAMP testing [13, 20] .

In conclusion this study shows that the variplex SARS-CoV-2 RT-LAMP assay may serve as an easy-to perform rapid molecular test to be combined with RT-PCR in order to ensure an efficient workflow of timely and accurate diagnosis even at times of high work load and increased testing requests. The major limitation of this work was the relatively small sample size due to low numbers of COVID-19 patients in our hospital. Future studies are needed to examine the utility of RT-LAMP under routine conditions with high sample throughput. 

The authors declare no conflicts of interest.

The authors declare no conflicts of interest.

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We thank Katrin Schützler, Sylvia Stoll, Beate Haschke, and Christina Gödicke for excellent technical assistance. The study was supported by internal funding.
