key: cord-0252432-emeh4byv authors: Mokhtar, Khelil Mohamed title: Improved COVID-19 Testing by Extraction-Free SARS-CoV-2 RT-PCR date: 2020-11-20 journal: EJIFCC DOI: nan sha: f8a5c4505f84fa91a5276d3cf3dad2756250fc05 doc_id: 252432 cord_uid: emeh4byv The RNA extraction is an important checkpoint for the detection of SARS-CoV-2 in swab samples, but it is a major barrier to available and rapid COVID-19 testing. In this study, we validated the extraction-free RT-qPCR method by heat-treatment as an accurate option to nucleic acid purification in Algerian population. Khelil Mohamed Mokhtar Improved COVID-19 testing by extraction-free SARS-CoV-2 RT-PCR Dear editor, The new emergence of the novel human coronavirus, in December 2019, in Wuhan City (China), rapidly evolved into a global pandemic. The virus was confirmed to have spread to Algeria in February 2020, which put notable pressure on public and private health laboratories as they attempt to keep up with demands for SARSCoV-2 testing despite shortage of reagents (1) . Currently, the widely used protocol for SARS-CoV-2 detection is RT-qPCR assay preceded by purification of viral RNA from patient sample, typically from nasopharyngeal (NP) swab as described by CDC and WHO (2) (3) (4) . However, nucleic acid purification step is not only laborious and time-consuming, but the additional steps requiring manual handling can result in experimental errors, especially false positive results due to specimen-to-specimen carryover (5) . To address this issue, recent attempts have been made to circumvent RNA extraction in COVID-19 testing by performing RT-qPCR directly on heat-treated subject samples (65°C for 30 min or 95°C for 10min) or directly loading patient swab medium into RT-PCR reaction mix. Using heat-treatment approach the sensitivity ranged from 92 to 96% and specificity from 93 to 100% (6) . Here, we tested the direct method of SARS-CoV-2 RT-qPCR on heat-treated (Hit-RT-PCR) nasopharyngeal swab samples and compared the results with RNA-extraction based RT-PCR results. This study was conducted at the clinical laboratory of Institut Pasteur of M'sila, Algeria. Nasopharyngeal swabs (NP) from patients with high likelihood for COVID-19 were collected by medical infectiologists and deposited in viral transport medium at different healthcare institution of the city of M'sila. Arrived to the laboratory, samples were stored at -20°C until extracted and tested within 72h. For routine analysis, RNA was extracted from 140 μL of NP samples using the QIAamp Viral RNA Mini kit. Reverse transcription and quantitative PCR were performed using the Biogerm® novel Coronavirus (2019-nCoV) nucleic acid kit following the manufacturer instructions: Total reactions of 25μl were obtained by mixing 20μl of master mix (primers and probe mix: ORF1ab, N and RNase P genes) and 5 μl of clinical sample to fill the reaction. The thermal cycling steps were: stage1: 50°C for 10 min, stage2: 95°C for 5 min, stage3: 95°C for 10 sec, 55°C for 40 sec, 40 cycles. The RT-qPCR was performed on a Rotor-Gen Q real time PCR machine (Qiagen®) using the Rotor-Gen Software v2.3. We initially aimed to validate heat-treatment method to get an accurate view of its performance in a real world clinical diagnostic setting. We blindly heated a panel of aliquots from 60 NP samples representing intermediate (CT of 20 -30) and low (CT of more than 30) viral RNA loads by direct RT-qPCR. The SARS-CoV-2 Ct levels (ORF1ab and N) in these samples were previously determined by RT-qPCR that included RNA extraction (Ct cutoff ≤38). NP swab samples were thermally treated in water bath at 65°C for 30 min. Samples were then placed in room temperature for 15 min, vortexed for 10 seconds, centrifuged at 1000g for 1 min and 5µl of the supernatant was directly loaded into RT-qPCR reaction. Comparably, aliquots from 161 NP samples were subjected to heat-treatment but with increasing heating time to 60 min. An agreement analysis (positive and negative percent agreement) were applied between diagnostic results of our experiment and results obtained by the conventional SARS-CoV-2 testing protocol. Diagnostic results were considered as categorical variables (1 for the presence of SARS cov2 infection and 0 for the absence of infection). All statistical analysis were performed using R version 3.6.0 (R Core Team, 2014) (7) . In this work we used anonymized material from Khelil Mohamed Mokhtar Improved COVID-19 testing by extraction-free SARS-CoV-2 RT-PCR samples that had been collected for clinical diagnostics of SARS-CoV-2. We found a weak agreement when NP samples were heated for 30min (PPA: 58%, 95%CI: 45 to 69%). But, the agreement increased (PPA: 78%, 95%CI: 70 to 84%) when we increased the heating time to (60 min). We also found a substantial agreement between N gene results of extracted and heat-inactivated samples (overall agreement 78%, 95%CI 70 to 83%) but a weak agreement for ORF1ab gene (overall agreement 45%, 95%CI 37 to 52%). Ct values of N gene for hit-RT-qPCR samples were higher than for RNA eluates of the same samples (mean difference =1.9 Ct). Surprisingly, three samples were identified as COVID-19 positive by 60 min heat-treatment RT-qPCR (one sample positive for N and ORF1ab and two for only N) but were negative on extracted RNA. Figure 1 and 2 show the full results of this experiment while Table 1 provides a summary. Clinical laboratories of the developing world are overwhelmed with COVID-19 testing demands. As a means to validate heat-treatment RT-PCR method in our clinical laboratory, we have shown that prior heating at 65°C for 30 min was less accurate compared to prior heating at 65°C for 60 min. Our observation were not corroborated by previous results which showed that prior heating at 65°C for 30 min was adequate to correctly identify 92 to 96% of screened samples. This could be explained by difference in the composition of viral transport medium used (Inhibitory agents from the swab and medium may inhibit RT-qPCR) or a mutations in the Algerian strain of SARS-cov2, rendering the virus more resistant to heat-treatment. Our improved protocol correctly identified 100% of clinical samples with viral load between (20 and 30 Ct). The only samples missed were those among lower Ct range (Ct> 30). Of the 2065 cases with a positive diagnosis at "Institut Pasteur of M'sila" by our clinical laboratory at the time of writing, only 27% would fall in this low Ct range, which demonstrate that our improved protocol will accurately detect the majority of COVID-19 patients. Evidence that analytical sensitivity of heat-treatment RT-PCR was inferior (higher Ct values) compared to extraction-based RT-qPCR is that heating for long time may degrade RNA in presence of metal ions and/or RNases and that more RNA was loaded for eluates compared to Hit-RT-PCR. Furthermore, the higher performance of primers and probes targeting short amplicon (N, 110 bp) confirmed previous reports. Hence, short amplicons targets may be more suitable for Hit-RT-qPCR protocol. A surprising finding was that heat-treatment RT-PCR identified three samples as COVID-19 positive while they had been identified as COVID-19 negative by conventional protocol. The Ct values of heat-treatment RT-PCR samples were high (> 30) suggesting one possible explanation of this phenomenon: NP samples may had very low viral RNA load that was below the limit of detection -i.e the lowest concentration level with a detection rate of 95% for positive resultsof the RT-PCR kit (1000 copies/ml) (9) . So, negative results in patients with typical symptoms of COVID-19 may become detectable by repeating the test. Unfortunately, we were unable to confirm COVID-19 positivity by collection of a new swab samples. In summary, we have shown that screening for SARS-CoV-2 infection by RT-qPCR could be achieved through heat-treatment protocol (65°C for 60 min) without the use of RNA extraction kits, in the studied population. We hypothesize that each clinical laboratory should validate its own heat-treatment protocol which may be specific to the pre-analytical (viral transport medium composition) and environmental factors. Previous reports suggest that initial negative result by heat-treatment RT-PCR should be repeated by RNA extraction for: symptomatic patients, healthcare personnel, and others with a high suspicion of COVID-19 (8) . However, Khelil Mohamed Mokhtar Improved COVID-19 testing by extraction-free SARS-CoV-2 RT-PCR Heat-map of ORF1ab and N Ct values for (65°C, 30min) protocol ID 65°C,30min Eluate Rnase P ORF1ab N ORF1ab N 1 38 39 29 27 24 2 41 41 28 28 24 3 41 41 35 31 31 4 41 41 35 31 25 5 38 36 26 25 31 6 41 35 27 26 24 7 41 41 32 30 27 8 41 41 30 28 24 9 41 41 30 29 25 10 41 41 28 26 28 11 41 41 27 26 27 12 24 22 41 22 28 13 41 41 29 28 25 14 32 28 25 24 24 15 34 33 31 30 25 16 41 38 35 32 25 17 41 37 33 32 27 18 41 37 36 32 30 19 35 33 30 28 26 20 41 41 36 33 27 21 41 35 32 30 27 22 35 37 35 35 30 23 25 23 23 22 25 24 41 41 30 33 24 25 26 23 23 22 24 26 28 25 25 24 23 27 34 27 26 25 25 28 35 32 31 29 29 29 36 36 32 35 26 30 41 37 36 33 30 31 35 33 39 34 24 32 41 41 35 33 26 33 41 41 37 35 27 34 41 41 36 36 25 35 23 24 22 23 26 36 33 32 28 28 30 37 37 35 33 32 29 38 41 41 35 34 24 39 29 28 23 24 25 40 41 41 41 39 29 41 41 41 36 35 26 42 33 32 28 28 25 43 30 29 25 26 25 44 41 41 36 37 26 45 29 29 26 26 28 46 41 41 37 35 8 27 23 23 23 24 9 26 22 23 23 25 10 41 31 30 30 28 11 30 24 24 24 27 12 29 23 24 24 28 13 41 33 33 32 25 14 37 30 28 29 24 15 41 41 41 41 25 16 41 41 41 41 25 17* 36 32 41 41 27 18 41 41 41 41 33 19* 41 34 41 41 26 20 41 31 41 36 27 21 41 32 31 32 27 22 32 27 41 37 30 23 41 41 31 31 25 24 41 30 27 27 24 25 41 34 35 33 24 26 28 23 23 23 23 27 27 29 28 28 25 28 41 36 33 33 29 29 41 30 25 23 26 30 41 36 33 33 30 31 41 41 41 41 24 32 41 39 41 41 26 33 30 28 26 26 27 34 41 41 41 41 25 35 41 41 41 36 26 36 41 41 41 37 30 37 41 41 41 41 29 38 41 30 28 28 24 39 41 41 41 38 25 40 41 37 41 37 29 41 41 30 29 29 26 Control for RNA degradation by RT-PCR for RNase P transcripts in the same samples is shown on the right. Three samples, marked with asterisk, negative in extraction-based routine diagnosis but positive by hit-RT-PCR. Khelil Mohamed Mokhtar Improved COVID-19 testing by extraction-free SARS-CoV-2 RT-PCR based on recent evidence showing the oddity of SARS-CoV-2 that can be cultured in respiratory samples 9 days after symptom onset, notably in patients with mild disease, it appears that retesting in such patients may not be necessary (10) . Such a strategy would drastically reduce the need for RNA extraction for a substantial portion of future COVID-19 tests. To stop COVID-19, test everyone, repeatedly Centers for Disease Control and Prevention & Division of Viral Diseases. CDC 2019-Novel Coronavirus Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Molecular Contamination and Amplification Product Inactivation Massive and rapid COVID-19 testing is feasible by extraction free SARS-CoV-2 RT-qPCR R: A language and environment for statistical computing. R Foundation for Statistical Computing Direct RT-qPCR detection of SARSCoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step Limits of Detection of 6 Approved RT-PCR Kits for the Novel SARS Coronavirus-2 (SARS-CoV-2) Brief: Criteria for releasing COVID-19 patients from isolation We are grateful to Mekki Oussama who participated in sample handling, organization and performed experiments. 36 33 29 28 24 79 41 41 41 41 25 80 41 41 41 36 28 81 41 31 31 30 27 82 41 33 36 34 28 83 41 33 37 35 25 84* 41 32 41 41 24 85 41 35 36 33 25 86 41 41 36 33 25 87 41 41 41 41 27 88 31 25 27 26 33 89 35 29 27 26 26 90 41 35 34 32 27 91 41 32 31 29 27 92 27 23 22 22 30 93 41 38 41 35 25 94 41 32 31 29 24 95 41 23 22 21 24 96 41 37 35 31 23 97 41 35 26 27 25 98 41 29 36 34 29 99 41 27 24 24 26 100 33 28 27 26 35 101 34 28 27 26 24 102 41 31 29 27 26 103 38 32 30 28 27 104 41 36 27 26 25 105 41 23 22 22 26 106 41 41 37 35 30 107 41 41 41 41 29 108 41 38 36 35 24 109 41 41 41 41 25 110 41 22 22 22 29 111 41 22 22 22 26 112 41 27 22 23 25 113 41 29 34 34 25 114 41 41 35 35 26 115 41 41 38 36 28 116 25 24 22 23 29 117 41 41 41 36 23 118 41 41 41 41 32 119 41 41 41 41 30 120 30 25 24 23 24 121 30 25 24 23 24 122 34 29 25 25 25 123 33 31 28 27 27 124 41 41 36 36 25 125 41 36 37 35 28 126 31 29 25 25 25 127 36 35 32 32 28 Khelil Mohamed Mokhtar Improved COVID-19 testing by extraction-free SARS-CoV-2 RT-PCR 128 34 31 28 28 25 129 36 32 29 28 23 130 41 37 41 38 32 131 41 37 33 33 30 132 37 35 29 28 24 133 41 34 30 30 23 134 28 26 23 23 25 135 31 29 25 25 33 136 41 41 35 33 30 137 41 41 34 32 30 138 32 29 23 22 30 139 41 41 32 33 31 140 41 41 33 32 25 141 41 41 28 30 24 142 41 41 33 32 24 143 27 25 21 21 31 144 32 29 24 24 25 145 41 41 34 35 31 146 41 41 35 34 24 147 38 34 30 30 27 148 41 41 30 31 24 149 41 41 30 33 25 150 38 31 25 25 28 151 39 34 30 30 27 152 41 36 29 29 28 153 38 29 24 24 25 154 41 41 30 33 24 155 41 41 31 32 25 156 41 41 35 35 25 157 41 41 26 35 27 158 41 41 35 33 33 159 41 41 35 34 26 160 27 25 21 21 27 161 28 26 23 23 27