Validation of new equipment for SARS-CoV-2 diagnosis in Ecuador: Detection of the virus and antibodies generated by disease and vaccines with one POC device

Abstract

The COVID-19 pandemic underscored the critical need to enhance screening capabilities and streamline diagnosis. Point-of-care (POC) tests offer a promising solution by decentralizing testing. We aimed to validate the PLUM device (LSK Technologies Inc.), a portable optical reader, to detect SARS-CoV-2 RNA using direct RT-LAMP targeting the ORF1a and E1 genes and patient antibodies by ELISA. The direct RT-LAMP assays employ nasopharyngeal swabs and bypass RNA extraction protocols through a brief chemical and physical lysis step. Test sensitivity and specificity were assessed against gold-standard detection methods in laboratory and field conditions. For samples with Ct values below 25, direct RT-LAMP showed 83% sensitivity and 90% specificity under laboratory conditions and 91% sensitivity and 92% specificity under field conditions. The nucleocapsid antigen antibody assay had 99% positive percent agreement (PPA) and 97% negative percent agreement (NPA), outperforming spike-RBD antigen (98% PPA, 92% NPA) and seroprevalence (98% PPA, 88% NPA) under laboratory conditions. Under field conditions, similar results were found for antibody detection for the nucleocapsid antigen (93% PPA; 100% NPA), spike-RBD (100% PPA; 94% NPA), and seroprevalence (90% PPA; 94% NPA). This study validated the PLUM device as a dual POC tool for direct RT-LAMP-based SARS-CoV-2 and ELISA-based COVID-19 antibody detection.

Fig 1. Distribution of nasopharyngeal samples positive for SARS-CoV-2.

Comparison of the Ct values between all positive samples (n= 720) and the positive samples selected for laboratory validation of RT-LAMP (n= 149). Mean ± SEM. Mann-Whitney U test (p > 0.05).

Fig 2. Direct RT-LAMP in the PLUM, validation under laboratory conditions.

(A) ROC curve evaluating the PLUM’s performance for detecting SARS-CoV-2 by direct RT-LAMP. (B) Distribution of RT-LAMP TTRs against RT-qPCR Ct values for 137 positive (red dots) and 144 negative (blue dots) nasopharyngeal samples. RT-qPCR and RT-LAMP positives were defined by Ct < 35.0 and TTR ≤ 40.9’, respectively (dotted lines). (C) Distribution of RT-LAMP and RT-qPCR values for 117 positive and 144 negative nasopharyngeal samples with Ct values below 30. (D) Distribution of RT-LAMP and RT-qPCR values for 96 positive and 144 negative nasopharyngeal samples with Ct values below 25. (E) ROC curve evaluating the PLUM’s performance for detecting internal control (IC) by direct RT-LAMP. (F) Distribution of RT-LAMP TTRs for IC from 137 positive and 144 negative samples as determined by RT-qPCR for SARS-CoV-2. Mean ± SEM. Mann-Whitney U test (p > 0.05).

Fig 3. Direct RT-LAMP in the PLUM, validation under field conditions.

(A) Distribution of RT-LAMP TTRs against RT-qPCR Ct values for 23 positive (red dots) and 26 negative (blue dots) nasopharyngeal samples. RT-qPCR and RT-LAMP positives were defined by Ct < 35.0 and TTR ≤ 40.9’, respectively (dotted lines). (B) Distribution of RT-LAMP TTRs for IC from 23 positive and 26 negative samples as determined by RT-qPCR for SARS-CoV-2. Mean ± SEM. Mann-Whitney U test (p > 0.05).

Fig 4. Detection of COVID-19 antibodies in sera samples by ELISA assays in the PLUM device, validation under laboratory conditions.

(A) Distribution of spike-RBD ELISA absorbance (ABS) relative ratio as determined by a microplate reader against PLUM reading units (PRU) relative ratio from 643 positive (red dots) and 153 negative (blue dots) sera samples. (B) Distribution of N ELISA ABS relative ratio against PRU relative ratio from 272 positive and 528 negative sera samples. (C) Distribution of seroprevalence results from the average ABS from spike-RBD and N against the average PRU from the same antigens using 175 positive and 125 negative sera samples. Cohen’s kappa statistics are shown. Dashed lines represent the threshold determined by ROC curve analysis (Table 3).

Fig 5. Detection of COVID-19 antibodies in sera samples by ELISA assays in the PLUM device, validation under field conditions.

(A) Distribution of spike-RBD ELISA absorbance (ABS) relative ratio as determined by a microplate reader against PLUM reading units (PRU) relative ratio from 44 positive (red dots) and 18 negative (blue dots) sera samples. (B) Distribution of N ELISA ABS relative ratio against PRU relative ratio from 14 positive and 48 negative sera samples. (C) Distribution of seroprevalence results from the average ABS from spike-RBD and N against the average PRU from the same antigens using nine positive and 18 negative sera samples. Cohen’s kappa statistics are shown. Dashed lines represent the threshold determined by ROC curve analysis (Table 3).