key: cord-0743430-bc34kg5c
authors: Bruno, Alfredo; de Mora, Domenica; Freire-Paspuel, Byron; Rodriguez, Angel S.; Paredes-Espinosa, Maria Belen; Olmedo, Maritza; Sanchez, Martha; Romero, Jennifer; Paez, Michelle; Gonzalez, Manuel; Orlando, Alberto; Garcia-Bereguiain, Miguel Angel
title: Analytical and clinical evaluation of a heat shock SARS-CoV-2 diagnosis method without RNA extraction for N and E genes RT-qPCR.
date: 2021-06-21
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.06.038
sha: badac7d0fb2304472684cd4d0db060585ddfea25
doc_id: 743430
cord_uid: bc34kg5c

BACKGROUND: : The COVID-19 pandemic has caused significant supply shortages worldwide for SARS-CoV-2 molecular diagnosis, like RNA extraction kits. OBJECTIVE: : The aim of our study was to evaluate the clinical performance and analytical sensitivity of a simple SARS-CoV-2 diagnosis protocol based on heat shock without RNA extraction using both "CDC" (N gene) and "Charite" (E gene) RT-qPCR protocols. RESULTS: : 1,036 nasopharyngeal samples, 543 of them SARS-CoV-2 positive, were analyzed. The heat shock method correctly identified 68,8% (232/337) and 89.4% (202/226) SARS-CoV-2 positive samples for N gene and E gene, respectively. Analytical sensitivity was assessed for heat shock method using the CDC RT-qPCR protocol, obtaining sensitivity values of 98,6%, 93,3% and 84,8% for limit of detection of 100.000, 50.000 and 20.000 viral RNA copies/mL of sample. CONCLUSIONS: : Our findings show that a simple heat shock SARS-CoV-2 RT-qPCR diagnosis method without RNA extraction is a reliable alternative for potentially infectious SARS-CoV-2 positive patients. This affordable protocol can help to overcome the cost and supply shortages for SARS-CoV-2 diagnosis, especially in developing countries. In Ecuador, it has been used already by laboratories in the public health system for more than 100.000 specimens.

The Humanity is facing the biggest public health crisis since the "Spanish flu" in 1918. The Coronaviruses Disease 2019 pandemic, caused by an infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has challenged public health systems worldwide since the initial outbreak in the Chinese city of Wuhan in December 2019. By January 31st 2021, SARS-CoV-2 had caused more than 100 million infections and 2.2 million deaths worldwide (https://coronavirus.jhu.edu/map.html).

The COVID-19 pandemic has challenged public health systems worldwide, not only for patient care and surveillance, but also to guarantee the quality and availability of SARS-CoV-2 related diagnosis tools. COVID-19 pandemic continues to be a worldwide public health concern and the diagnostic improvements for successful case detection, contact tracing and control of the spread of SARS-CoV-2 infection is are still a challenge after more than 10 months of the outbreak (CDC, 2020a; Freire Paspuel et al. 2020a; Freire Paspuel et al. 2020b) . Supply shortage of SARS-CoV-2 testing materials have forced a narrow testing strategy focused on the care of hospitalized patients, hampering efforts to identify and prevent community transmission of SARS-CoV-2, not only on in developing countries like Ecuador but even on in USA (Kavanagh et al. 2020; Schneider et al. 2020) . Although there are several SARS-CoV-2 diagnostic tools available in the market such as RT-LAMP, RNA extraction free systems like Cepheid Xpert Xpress SARS-CoV-2 assay or rapid antigen tests, standard RT-qPCR assays with a previous RNA extraction step remains the gold standard after more than one year of COVID-19 pandemic.

Organization (WHO) both still recommend strict RT-qPCR SARS-CoV-2 molecular diagnostic protocols with specifications for sample collection and RNA extraction (CDC 2020b; WHO 2020) .

Under this scenario, RNA extraction kits are among the most highly demanded supplies for SARS-CoV-2 diagnosis. A Few reports have already described a potential solution to overcome RNA extraction kits dependency by using simple heat inactivation and extraction step as an alternative to automated RNA extraction kit-based systems, which are also more expensive and time and labor demanding (Barza et al. 2020; Fomsgaard et a al. 2020; Wing-Ho Chu et al. 2020) . The heat shock principle is based on the disruption of the physical integrity of viruses at high temperatures, allowing the release of viral RNA for RT-PCR detection. However, those reports differ on the sensitivity associated to the heat shock protocol and the comparison is mostly made with automatized magnetic beads RNA extraction The present study evaluated a heat shock method for SARS-CoV-2 diagnosis without RNA extraction using the CDC (N gene) and Charite (E gene) RT-PCR protocols (Corman et at. 2020; Lu et al. 2020) , with significant sample size. We compared the clinical performance and analytical sensitivity of the heat shock method for detection of SARS-CoV-2 to results obtained using a column based manual RNA extraction kit protocol.

Study design. a total number of 1,036 nasopharyngeal swabs collected on 0.5mL TE pH 8 buffer were included on in this study, coming from two different laboratories in Guayaquil ("Instituto Nacional de Salud Pública e Investigación Leopoldo Izquieta Pérez": "INSPI") and Quito ("Universidad de Las Américas": "UDLA"), as it is detailed in Figure 1 . Also, negative controls (TE pH 8 buffer) were included as control for carryover contamination, one for each set of RNA Table 3 and 4). RNase P as an RNA extraction quality control (Lu et al. 2020 ). These samples were processed at the laboratory of "UDLA" located in Quito. We referred along the text to this SARS-CoV-2 RT-PCR protocol as the "CDC protocol". LightCycler 480 II system Roche instrument were was used for RNA extraction and thermal cycling. Briefly, the "Charité University-Berlin Institute of Virology" (Berlin, Germany) designed

LightMix SarbecoV E-gene plus EAV control kit is based on E gene detection probe for SARS-CoV-2 and EAV as an RNA extraction quality control (Corman et al. 2020 ). These samples were processed at the laboratory of "INSPI" located in Guayaquil. We referred along the text to this SARS-CoV-2 RT-PCR protocol as de "Charite protocol".

RT-qPCR for SARS-CoV-2 diagnosis using heat shock method without RNA extraction. All the samples processed by the "CDC" and "Charite" protocols using RNA extraction kits were also processed using our heat shock method without RNA extraction. The remaining volume (approximately 300 uL) of transport mediums after RNA extraction (200 uL) was used for the heat shock method. The samples were centrifuged for 1 min up to 14,000 rpm, supernatant was carefully removed and 50uL of RNase free water was added, followed by vortexing for 30 seconds. Samples were then placed on 0.2mL tubes at a thermal cycler. A heat shock of 99 °C for 5 minutes was applied, followed by cooling at 4°C for 5 minutes. To avoid RNA degradation, samples were processed for RNA extraction and heat shock method within the same day.

Analytical Sensitivity. Limit of detection (LoD) thresholds for sensitivity calculations were addressed by calculating viral loads of the samples processed by the "CDC" protocol. The 2019-nCoV N positive control (IDT, USA), provided at 200.000 genome equivalents/mL was used for calibration curves to obtain the viral loads of the samples. Viral loads can be expressed as copies/uL of RNA extraction or copies/mL of sample; the conversion factor is 200, as 0.2mL of sample is used for RNA extraction and 40uL is used as final elution volume of RNA extraction.

Ethics statement. All samples have been submitted for routine patient care and diagnostics.

Ethics approval was not sought because the study involves laboratory validation of test methods and the secondary use of anonymous pathological specimens that falls under the c y ' x p ' y "I u N c S u Pú c I v ación Leopoldo Izquieta Pérez" review board and "Comité de Etica para Investigación en Seres Humanos" from "Universidad de Las Américas".

Clinical performance for heat shock SARS-CoV-2 RT-PCR diagnosis method without RNA extraction for preselected SARS-CoV-2 positive samples.

150 samples SARS-CoV-2 positive samples with RNA extraction were tested following the "Charite protocol" for the heat shock method. 133 out of 150 SARS-CoV-2 positive samples were also positive for the heat socked shock method, resulting a sensitivity of 88.7% (Table 1) . 

The total number of 1,036 samples were analyzed for both standard RNA extraction and heat shock method. 543 samples were positive for RNA extraction-RT-qPCR, and 434 of them were also positive for heat shock-RT-PCR, yielding an overall sensitivity for the heat shock method of 77.1% (434/563; see Table 4 ).

For the Charité protocol, a total number of 566 samples were analyzed for both standard RNA extraction and heat shock method. 226 samples were positive for RNA extraction-RT-qPCR, and 202 of them were also positive for heat shock-RT-PCR, yielding an overall sensitivity for the heat shock method followed by RT-PCR for E gene of 89.4% (202/226).

For the CDC protocol, a total number of 470 samples were analyzed for both standard RNA extraction and heat shock method. 337 samples were positive for RNA extraction-RT-qPCR, and shock method followed by RT-PCR for N gene of 68.8% (232/337).

Our results support that the direct detection of SARS-CoV-2 using a heat shock method based on a 99 C heat inactivation and release step for 5 minutes without RNA extraction is a reliable alternative to the use of column based manual RNA extraction kits. We observed a significant switch towards higher Ct values for E, N1 and RNaseP amplicons using the heat shock method. We performed the RT-PCR with the same amount of sample no matter regardless whether heat shock of RNA extraction was performed,. and Also, similar volume was used for heat shock (remaining volume of 300uL) or RNA extraction (200 uL). So, the lack of sensitivity observed is probably due to the less efficient RNA concentration by centrifugation and elution on a smaller volume, compared to the use of ionic binding columns like the ones included on RNA extraction kits. Additionally, the sensitivity for the heat shock method was better for E gene based RT-PCR (89.4%) than for N gene (68.8%), but because each of the RT-PCR protocol were developed at a different laboratory, we cannot completely rule out that those differences may be not associated to the gene targets but to experimental variability. (Hasan et al. 2020) and 150 (Bruce et al. 2020) . Also, our study is the only one using two of the most worldwide used RT-PCR protocol for SARS-CoV-2 diagnosis, like "CDC and Charite protocols" (CDC 2020b; Corman et al. 2020; Lu et al. 2020 ). Nevertheless, we found and overall sensitivity for our heat shock method of 77.5%; similar values of sensitivity ranging from 58% to 97.4% has been reported on previous studies (Barza uc -Ho Chu et al. 2020), as it is summarized on Table 4 .

The previous publications addressing heat shock methods without RNA extraction for SARS-CoV-2 diagnosis clearly indicated that failure to detect SARS-CoV-2 positive samples happened for high Ct values in the range of 32 to 40 ( uc -Ho Chu et al. 2020) . However, only one of those reports address the sensitivity of the heat shock methods in terms of viral load, reporting a sensitivity of 95% for samples above 66.000 viral copies/mL (Hasan et al. 2020) . Although the main limitation of that study is the reduced sample size, only 18 SARS-CoV-2 positive samples (Hasan et al. 2020) , those results are on agreement with the sensitivity of 93.3% for viral loads bigger than 50.000 copies/mL that we obtained.

It is important to notice that our heat shock SARS-CoV-2 RT-PCR method has a sensitivity up to 98.6% for viral loads bigger than 100.000 copies/mL. Considering the viral loads frequency distribution for SARS-CoV-2, this high LoD would potentially exclude more than 30% of true positive cases (Kleiboeker et al. 2020; Lavezzo et al. 2020 ). It has also been recently reported that only patients with viral loads over 1 million copies/mL would be infectious ( 2020) Considering the worldwide high demand of reagents for SARS-CoV RT-qPCR diagnosis, supply shortages is a fact, hampering extensive testing at developing countries like Ecuador. Under this scenario, SARS-CoV-2 diagnosis methods alleviating cost and dependency on supplies are crucial to increase SARS-CoV-2 diagnosis capacity and even to avoid diagnosis disruption. This was the case with the SARS-CoV-2 National Reference Laboratory at "Instituto Nacional de Salud Pública Leopoldo Izquieta Pérez" in Ecuador, where the method described on this study has been used for more than 100.000 samples at the time that RNA extraction kits were not available in the country.

Funding.

This study was partially funded by Universidad de Las Americas (Quito, Ecuador).

Authorship contribution statement.

All authors contributed to study conceptualization, experimental procedures and revision and approval of final version of the manuscript.

Alfredo Bruno, Byron Freire-Paspuel, Domenica de Mora performed most of the experiments and analyzed the data. Miguel Angel Garcia Bereguiain wrote the manuscript.

Declaration of Competing Interests.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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

An alternative work flow for molecular detection of SARS-CoV-2 -escape from the NA extraction kit-shortage

One Health" inspired SARS-CoV-2 surveillance: The Galapagos Islands experience

Cotton-Tipped Plastic Swabs for SARS-CoV-2 RT-qPCR Diagnosis to Prevent Supply Shortages

Evaluation of nCoV-QS (MiCo BioMed) for RT-qPCR Detection of SARS-CoV-2 From Nasopharyngeal Samples Using CDC FDA EUA qPCR Kit as a Gold Standard: An Example of the Need of Validation Studies

Sample pooling of RNA extracts to speed up SARS-CoV-2 diagnosis using CDC FDA EUA RT-qPCR kit

Analytical sensitivity and clinical performance of a triplex RT-qPCR assay using CDC N1, N2 and RP targets for SARS-CoV-2 diagnosis

Low Clinical Performance of "Isopollo COVID19 detection kit" (Monitor, South Korea) for RT-LAMP SARS-CoV-2 diagnosis: a call for action against low quality products for developing countries

RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA

Access to lifesaving medical resources for African countries: COVID-19 testing and response, ethics, and politics

SARS-CoV-2 Viral load Assessment in Respiratory Samples

Suppression of a SARS-CoV-u I u c p y V ' Nature

US CDC Real-Time Reverse Transcription 20.000 copies / mL

We thank "Ministerio de Salud Pública" for their support for SARS-CoV-2 diagnosis at "Instituto Nacional de Salud Pública e Investigación Leopoldo Izquieta Pérez". We also thank Dr Tannya Lozada from "Dirección General de Investigación de la Universidad de Las Américas", and the authorities from "Universidad de Las Américas", for logistic support to make SARS-CoV-2 diagnosis possible at our research laboratories.References.

and RNaseP gene and viral loads for SARS-CoV-2 random samples with heat shock method and RNA extraction method. Viral loads are detailed on copies/uL of RNA extraction solution. A 200X conversion factor is used to transform these values to copies/mL of sample (PP means presumptive positive samples: only amplification for one viral gene target).