Diagnostic performance of a colorimetric RT -LAMP for the identification of SARS-CoV-2: A multicenter prospective clinical evaluation in sub-Saharan Africa

Abstract

Background: Management and control of the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus SARS-CoV-2 is critically dependent on quick and reliable identification of the virus in clinical specimens. Detection of viral RNA by a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a simple, reliable and cost-effective assay, deployable in resource-limited settings (RLS). Our objective was to evaluate the intrinsic and extrinsic performances of RT-LAMP in RLS.

Methods: This is a multicenter prospective observational study of diagnostic accuracy, conducted from October 2020 to February 2021 in four African Countries: Cameroon, Ethiopia, Kenya and Nigeria; and in Italy. We enroled 1657 individuals who were either COVID-19 suspect cases, or asymptomatic and presented for screening. RNA extracted from pharyngeal swabs was tested in parallel by a colorimetric RT-LAMP and by a standard real time polymerase chain reaction (RT-PCR).

Findings: The sensitivity and specificity of index RT LAMP compared to standard RT-PCR on 1657 prospective specimens from infected individuals was determined. For a subset of 1292 specimens, which underwent exactly the same procedures in different countries, we obtained very high specificity (98%) and positive predictive value (PPV = 99%), while the sensitivity was 87%, with a negative predictive value NPV = 70%, Stratification of RT-PCR data showed superior sensitivity achieved with an RT-PCR cycle threshold (Ct) below 35 (97%), which decreased to 60% above 35.

Interpretation: In this field trial, RT-LAMP appears to be a reliable assay, comparable to RT-PCR, particularly with medium-high viral loads (Ct < 35). Hence, RT-LAMP can be deployed in RLS for timely management and prevention of COVID-19, without compromising the quality of output.

Fig. 1

Stratification of sensitivities on RT-PCR Ct values. Stratification of the RT LAMP colorimetric results (positive/negative) on the RT-PCR (Liferiver) Ct values. The RT LAMP is a naked-eye colorimetric assay where the reaction incubated at 65 °C for 30 min turns from red (negative) to yellow (positive). Aggregated data from Italy, Ethiopia, Kenya and Nigeria are shown as yellow (positive) and red (negative) bars. Data for sensitivity within the various stratification windows are described in Table 2. To better correlate Ct values with SARS-CoV-2 RNA genomes a standard curve was build and used to interpolate values from RT-PCR. With this method 25 Ct correspond to 5.9 × 10⁶ (95% CI, 6.8 × 10⁶ – 5.2 × 10⁶) SARS-CoV-2 genomes/mL; 30 Ct correspond to 2.2 × 10⁵ (95% CI, 2.4 × 10⁵ – 1.8 × 10⁵) genomes/mL; 35 Ct correspond to 8.2 × 10³ (95% CI, 9.6 × 10³ – 7.0 × 10³) genomes/mL. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).