Due to its high sensitivity and specificity, RT-PCR is considered the gold standard for the detection of SARS-CoV-26,7. However, to maintain these very good performance qualities, this technology requires a sound laboratory infrastructure, trained professionals, and expensive equipment and facilities, which limits the accessibility to PCR testing, particularly in developing countries8,19. From this aspect, LAMP appears to be a promising alternative to PCR, as it can be implemented as a PoC method, with equipment costs much lower compared to PCR8,10,17,20. However, an improvement of the diagnostic sensitivity and specificity is required to be comparable with RT-PCR.
In an emergency diagnostic situation, as observed in the recent SARS-CoV-2 pandemic, a short sample-to-result time is essential for successful epidemiological disease management. As RT-PCR approaches usually take between 3–6 h for analysis (excluding transport time, pre-analytics, and reporting), one of the main advantages of POCT is that results are provided within minutes compared to several hours or days (see Table 3).
Although isothermal nucleic acid amplification and LAMP are comparable to RAT in terms of simplicity, convenience, and rapidity, their sensitivity and specificity are far more superior19,21,22. Based on our experience and according to various studies, false negative RAT results have been observed in samples with a low viral load, particularly in samples with a CT > 2523,24,25. In a pandemic setting, this may represent a burden as contact persons desire rapid results to exclude potential infection. The application of RATs at a very early stage after contact may reveal false negative results. Therefore, the is a high demand for more sensitive rapid diagnostic tests. A relatively high number of different isothermal nucleic acid amplification techniques have been developed and described, including but not limited to exponential amplification reactions (EXPAR), exponential rolling circle amplification (E-RCA), exponential strand displacement amplification (E-SDA), helicase-dependent amplification (HDA), nucleic acid sequence-based amplification (NASBA), and recombinase polymerase amplification (RPA)9,26,27,28,29,30. However, these techniques still have several shortcomings, with non-specific amplifications representing the largest hindrance for their rapid implementation in routine use as discussed elsewhere19,31,32. New LAMP-PoC devices are under continuous development and require clinical evaluation. The diagnostic accuracy of the recently described RHAM technology19, which uses an RNase HII reporter for signal visualisation, had not yet been investigated.
The present study is the first to report on the diagnostic sensitivity and accuracy of the RHAM technology in a PoC device (Pluslife Mini Dock) in a direct comparison to two well-established POC platforms (Abbott ID Now™ and Cepheid GeneXpert® IV), and additional performance characteristics of the Pluslife Mini Dock for the detection of SARS-CoV-2 variants. For this study, previously collected nasopharyngeal and oropharyngeal swab samples stored in UTM or PBS at -80 °C were used, which is, however, not in accordance with the original instructions for sample collection by the manufacturers for all three tests. The foreseen sample for these kits is freshly collected dry swab samples. For example, the instructions for Abbott ID Now™ COVID-19 state “direct test performance without elution into a VTM”, whereas the Cepheid Xpert® Xpress SARS-CoV-2 assay also accepts samples stored in VTM (3 mL, Copan or similar) for a maximum 8 h at RT or a maximum of 7 days at 2–8 °C in NaCl solutions. Yet, 98.7% of the samples used in the comparative accuracy study (study site 1) demonstrated valid results. This enables the acquisition of information regarding their diagnostic accuracy. With only one invalid result, the Pluslife Mini Dock was observed to be most stable in terms of specimen variability, with 99.7% of samples examined at study site 1 demonstrating valid test results. In contrast, Abbott ID Now™ showed a total of 31 invalid results, whereas Cepheid Xpert® Xpress had four invalid results. It cannot be excluded that the use of VTM may lead to reduced test accuracy in Abbott ID Now™. However, a previously published comparison between the collection method and sample type using Abbott ID Now™ COVID-19 and Cepheid Xpert® Xpress showed that approximately one-third of the samples that tested positive by the Cepheid Xpert® Xpress were negative using Abbott ID Now™ when using nasopharyngeal swabs in VTM compared to 45% when using dry nasal swabs33. This may indicate that Abbott ID Now™ is more prone to invalid results, regardless of the sample type used. The use of retrospective samples in this evaluation study, however, represents a limitation.
This may also be represented by the fact that all three PoC devices applied in this study demonstrate a very good diagnostic PPA of ≥ 99.00%. Interestingly, sample ID 018 (CT of 33.27) demonstrated ambiguous results upon measurement in all three devices. False negative results were observed with this sample using the Pluslife and Cepheid devices and an invalid result was observed with Abbott ID Now™, suggesting the presence of interfering substances in this particular sample. Of note, the sample was not excluded due to the uncertainty of this discrepancy and was retained in statistical analyses.
Despite the observation of 10% invalid results with Abbott ID Now™ (primarily in the negative cohort), the determined PPA of 100% is not in accordance to the literature. Sensitivities of 89.9% or even 79% (in comparison to 99% for Cepheid GeneXpert) were reported elsewhere34,35. However, different comparative studies regarding the PoC detection of SARS-CoV-2 show highly variable results. The PPA for Abbott ID Now™ range between 48 and 96% and for Cepheid GeneXpert® between 95 and 100%, with the studies demonstrating variable study designs and sample types20,35. In the present study, the PPA for the Pluslife Mini Dock was found to be 99.00%, for Abbott ID Now™ 100.00%, and for Cepheid GeneXpert® 98.99%, with a NPA of 100.00%, 98.90% and 94.09%, respectively.
Notable differences in the NPA were observed between the three PoC devices examined. Using 219 confirmed RT-PCR negative samples, the NPA was 100.00% for Pluslife. Abbott ID Now™ demonstrated an NPA of 98.90%, with two false positive cases. With 13 false positive cases, Cepheid GeneXpert® IV demonstrated the lowest NPA (93.72%) among all three PoC devices. The best analytical accuracy was observed for the Pluslife Mini Dock RHAM-based technology (99.68%), followed by Abbott ID Now™ (99.29%), and Cepheid GeneXpert® IV (95.42%).
One aspect that may reduce test accuracy regardless of RT-PCR or LAMP-based technologies are mutations in the target primer or probe-binding site. SARS-CoV-2 is highly susceptible to mutations in its RNA genome, with different subvariants emerging since its discovery in 201936,37. To further investigate the clinical accuracy of the Pluslife PoC device in identifying SARS-CoV-2 variants, 137 SARS-CoV-2 positive samples with known variants were investigated. With a mean time-to-positive result of 15–20 min, the Pluslife test demonstrated a sound PPA for all SARS-CoV-2 variants, ranging from Delta/Omicron (BA.1) in early 2022 to the current predominant variant XBB.1. There was only one false negative sample amongst the 40 XBB.1 samples analysed, with a relatively low CT value (CT = 31.79). With a PPA of 99.27% (136/137) and an NPA of 100.00% (104/104), the diagnostic accuracy for Pluslife determined by study site 1 was reproduced and confirmed by study site 2, representing two independent clinical evaluation studies.
Although there was no significant difference in the CT values across all SARS-CoV-2 variants (One-way ANOVA, P > 0.05), we observed a significant variation in the time-to-positive result across the SARS-CoV-2 variants (P < 0.0001). The mean test duration of XBB was significantly higher compared to most other variants (Mann Whitney U test; Delta, P < 0.0001; BA.1, P = 0.0055; BA.2, P = 0.9210; BA.4, P = 0.1566; BA.5, P = 0.0083), suggesting that the reaction for XBB.1 detection is somewhat slower. The cause of this deviation remains unclear. According to the manufacturer (Pluslife), in-silico analyses confirmed test accuracy, whereby the primers used were aligned to target regions to the different variants of concern (including XBB), suggesting that the test performance of the Pluslife Mini Dock for SARS-CoV-2 is not impaired by any known mutations in the SARS-CoV-2 variants. This observation underlines the importance of post-market surveillance for continued monitoring of clinical accuracy in viral diagnostics.
CT values are a valuable diagnostic tool for monitoring the course of an infection and can be correlated with the viral load, providing a semi-quantitative measure of the degree of infection, early onset of infection in asymptomatic contact persons, and for monitoring recovery progress in severe, hospitalised patients during the SARS-CoV-2 pandemic7,38,39. A similar “quantitative measure” quality would be appreciated from a POCT. As the reaction stops when the fluorescence signal exceeds the threshold, a correlation between time-to-result and viral load may exist. Therefore, we investigated whether a correlation existed between the time-to-result of the Pluslife Mini Dock and the CT value. However, no correlation was found (Pearson r = 0.01427, P = 0.8695, n = 135). Thus, the RHAM technology only indicated accurately the presence or absence of a viral infection but did not facilitate any significant (semi-) quantitative measurements.
In conclusion, the RHAM technology applied by the Pluslife Mini Dock PoC device demonstrated very good analytical PPA, NPA, and accuracy in identifying SARS-CoV-2 variants compared to RT-PCR results. The major advantage of PoC devices is that special laboratory facilities and trained personnel are not required. This facilitates the decentralised, reliable detection of pathogens of interest directly in a PoC setting. A POCT may be a preferred and better option for many developing countries that lack the necessary infrastructure and facilities. The RHAM technology is an emerging method poised to become of increasing relevance and application in preventing and controlling not only SARS-CoV-2 infections, but also future outbreaks of infectious diseases.
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- Source: https://www.nature.com/articles/s41598-024-64406-9