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. 2024 Aug 23:11:1445142.
doi: 10.3389/fmolb.2024.1445142. eCollection 2024.

Cross comparison of alternative diagnostic protocols including substitution to the clinical sample, RNA extraction method and nucleic acid amplification technology for COVID-19 diagnosis

Ismael Segura-Ulate  1 , Navilla Apú  1 , Bernal Cortés  2 , Jordi Querol-Audi  3   4 , Yamitzel Zaldívar  5 , Carlos Alexander Ortega  6 , Fernando Flores-Mora  1 , Andrés Gatica-Arias  1   7 , Germán Madrigal-Redondo  1
Affiliations

Cross comparison of alternative diagnostic protocols including substitution to the clinical sample, RNA extraction method and nucleic acid amplification technology for COVID-19 diagnosis

Ismael Segura-Ulate et al. Front Mol Biosci. .

Abstract

Background: the gold-standard diagnostic protocol (GSDP) for COVID-19 consists of a nasopharyngeal swab (NPS) sample processed through traditional RNA extraction (TRE) and amplified with retrotranscription quantitative polymerase chain reaction (RT-qPCR). Multiple alternatives were developed to decrease time/cost of GSDP, including alternative clinical samples, RNA extraction methods and nucleic acid amplification. Thus, we carried out a cross comparison of various alternatives methods against GSDP and each other.

Methods: we tested alternative diagnostic methods using saliva, heat-induced RNA release (HIRR) and a colorimetric retrotranscription loop-mediated isothermal amplification (RT-LAMP) as substitutions to the GSDP.

Results: RT-LAMP using NPS processed by TRE showed high sensitivity (96%) and specificity (97%), closely matching GSDP. When saliva was processed by TRE and amplified with both RT-LAMP and RT-qPCR, RT-LAMP yielded high diagnostic parameters (88%-96% sensitivity and 95%-100% specificity) compared to RT-qPCR. Nonetheless, when saliva processed by TRE and detected by RT-LAMP was compared against the GSDP, the resulting diagnostic values for sensitivity (78%) and specificity (87%) were somewhat high but still short of those of the GSDP. Finally, saliva processed with HIRR and detected via RT-LAMP was the simplest and fastest method, but its sensitivity against GSDP was too low (56%) for any clinical application. Also, in this last method, the acidity of a large percentage of saliva samples (9%-22%) affected the pH-sensitive colorimetric indicator used in the test, requiring the exclusion of these acidic samples or an extra step for pH correction.

Discussion: our comparison shows that RT-LAMP technology has diagnostic performance on par with RT-qPCR; likewise, saliva offers the same diagnostic functionality as NPS when subjected to a TRE method. Nonetheless, use of direct saliva after a HIRR and detected with RT-LAMP does not produce an acceptable diagnostic performance.

Keywords: COVID-19; RT-LAMP; SARS-CoV-2; alternative protocol; saliva sample.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic illustration of the accepted or traditional parts of the GSDP and the alternatives to each of these parts that are assessed in this study. Colored and dashed arrows show the combinations resulting in the different diagnostic protocols included in this comparison. GSDP, gold-standard diagnostic protocol; NPS, nasopharyngeal swab; TRE, traditional RNA extraction; HIRR, heat-induced RNA release; NAA, nucleic acid amplification; RT-LAMP, retrotranscription loop-mediated isothermal amplification; RT-qPCR, retrotranscription quantitative polymerase chain reaction.
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