Real-world evaluation of the Lucira Check-It COVID-19 loop-mediated amplification (LAMP) test

ABSTRACT In hospitals during the COVID-19 pandemic, laboratory testing was important to reduce SARS-CoV-2 transmissions, particularly for high-risk settings like the emergency department and pre-operative settings and for the safe return to work of exposed healthcare workers (HCWs). For these applications, delayed test results from laboratory nucleic acid amplification tests (NAATs) posed a barrier to maximizing efficient patient flow and minimizing staffing shortages. This quality improvement project sought to evaluate the performance of the Lucira Check-It COVID-19 Test, a rapid diagnostic test that used NAAT technology (NAAT-RDT). Using 10-fold serial dilutions of SARS-CoV-2, the analytical sensitivity of the NAAT-RDT was assessed against standard NAATs used for routine diagnostic testing. Clinical performance was assessed at two Nova Scotia hospitals in 405 cases with paired swabs tested by NAAT-RDT and laboratory-based NAATs. These represented three distinct populations: patients presenting to the emergency department (n = 208), patients in the pre-operative setting (n = 158), and patients presenting to community testing sites (n = 38). The analytical sensitivity of the NAAT-RDT and other laboratory NAATs was comparable. During clinical evaluation, the overall sensitivity and specificity were 92.9% and 98.3%, respectively, with little variation between settings. The Lucira NAAT-RDT is a portable and self-contained device that provides an easily interpreted result within 30 minutes following a bilateral nasal swab collection. Its performance was shown to be acceptable for use in three settings in this quality improvement project, facilitating patient flow and management. IMPORTANCE In hospitals during the COVID-19 pandemic, laboratory testing was important to reduce SARS-CoV-2 transmissions, while facilitating patient flow in the emergency department and pre-operative settings, and allowing for the safe return to work of exposed healthcare workers. Delayed test results from laboratory nucleic acid amplification tests (NAATs) posed a barrier to maximizing efficient patient flow and minimizing staffing shortages. This quality improvement project sought to evaluate the analytical and clinical performance of the Lucira Check-It COVID-19 Test, a point-of-care test that used NAAT technology, in the perioperative setting, emergency department, and community testing sites. We found the Lucira Check-It to have comparable performance to laboratory NAATs. It can be employed with little training for specimen collection, processing, and interpretation, and at a cost justifiable from the resources saved from avoiding sample transport and laboratory testing.

Comment 1: • The authors should provide an explana on in Table 1 for the absence of determined sensi vity in the periopera ve diagnos c test and possibly include the PPV as well.
• I am unsure about the meaning of "periopera ve" as used by the authors.Using the term "preopera ve screening" might be more appropriate.
• The sensi vity of the diagnos c tests in the emergency department was not sufficiently high.This aspect requires construc ve cri cism and a thorough examina on of its limita ons.
• The authors asserted that the majority of pa ents from both the pre-opera ve screening and emergency condi ons were asymptoma c (lacking respiratory symptoms).If the authors concur, they should provide the ra onale behind this observa on and the pretest likelihood ra o and engage in further discussion on this ma er.
• Table 1 should include the PPV and NPV values.
Comment 2: The authors should explain the sample size calcula on for all se ngs, but not for only one of periopera ve, ED, and community cases.Addi onally, to improve clarity, all se ngs groups in Table 1 should be displayed in the first row of the table.
Comment 3: Both the abstract and manuscript should be wri en concisely using well-refined English to enhance overall readability and quality.
Comment 4: The authors should offer clear defini ons of NAAT (Nucleic Acid Amplifica on Test) and NAAT-RDT (Nucleic Acid Amplifica on Test -Rapid Diagnos c Test) and consistently use these defined terms throughout the en re manuscript.
Comment 5: The authors should provide a comprehensive defini on of asymptoma c close contacts of COVID-19 cases and elucidate the reasons why evalua ng these pa ents is essen al for the study's objec ves.
Comment 6: The authors should provide informa on on the Cycle threshold (Ct) value of NAAT in true posi ve (TP) and true nega ve (TN) results of NAAT-RDT for be er understanding and analysis.
Comment 7: Regarding the claim of real-world tes ng, it is essen al for the authors to specify the total number of both symptoma c and asymptoma c pa ents who underwent NAAT-RDT tes ng to provide a clearer context for the study's applicability.
Comment 8: The implementa on of this study tle seems to focus on asymptoma c individuals rather than asymptoma c individuals suspected to have COVID-19.However, it would be beneficial if the authors could present more data regarding the sensi vity and specificity in symptoma c COVID-19 cases.These data should be cited in the introduc on and appropriately referenced.

Comment 9:
In Line 130, the authors men oned that the other two false nega ve results from the Lucira NAAT-RDT had paired specimens with low Ct values of 18.4 and 18.9, indica ng high viral loads during the infec ous period of SARS-CoV-2.The authors should elaborate on why nega ve NAAT-RDT results can occur during a period of high viral load to provide a more comprehensive understanding of the test's performance under such circumstances.

Comment 10:
In Line 134, the authors suggest that false nega ve Lucira results may be due to diagnos c failures resul ng from sequence mismatches between the Lucira target and circula ng SARS-CoV-2.However, to provide a more comprehensive understanding, the introduc on should include addi onal details about the basis and principles of the Lucira test, par cularly regarding the gene detec on part before discussing the possibility of needing to resort to sequencing.

Comment 11:
In Line 137, the authors acknowledge that false posi ve (FP) and false nega ve (FN) results can occur with any diagnos c test.It is important to note that while these occurrences are possible for any test, for nucleic acid amplifica on tests (NAATs) in general, higher sensi vity and specificity are usually expected, o en nearing 100%.

Reviewer 1 Authors evaluated Lucira COVID-19 LAMP test performance in hospital and clinic se ngs compared to standard NAAT. Though the results look promising, the paper miss lots of data (only 1 table included).
Supplementary Table S1 that contained relevant data was not cited in the body of the text.We apologize for this oversight.Table S1 has been moved from the supplementary material to the body of the manuscript to make it more visible to readers and we have re-labelled it Table 1 (and re-labelled the previous Table 1 as Table 2 accordingly).Unfortunately, we are limited beyond this by two figure/table limita ons of the "Observa ons" ar cle type.
In Background, authors should expand NAAT-based RDTs; as far as I know, there are diverse rapid methods for SARS-CoV-2 tes ng, such as ID NOW, LAMP-CRISPR, SDA, RPA.Authors should also give some background of Lucira, e.g.mechanism of the assay, is the readout colorimetric or fluorescence?
We have modified the background to recognize the other isothermal technologies and indicated the rationale of why Lucira was chosen.We have also added more to the description of the technology used with Lucira, a RT-LAMP targeting N and Orf7b/8 genes with a colorimetric output detected by change in pH during target amplification.See lines 73-76.
Can authors elaborate how many samples were tested by each of Cobas, Xpert and Altima platforms, were all 405 samples tested on these three qPCR assays to derive concordance?We know there are variations of performance between platforms.Maybe a table should be included to describe how concordance of TP and TN is obtained.NAAT results reported to the ordering physician did not dis nguish which diagnos c test was used and therefore, the number for each NAAT was not possible to collect for this quality ini a ve.The Lucira results were compared to one of three possible commercial NAATs used in clinical laboratories.This has been clarified in the text.How the NAAT results were used to define TP and TN Lucira was defined in the methods.We have added modifica ons to clarify the methods.See lines 103-112.
Authors should include a table or figure showing how the serial dilu ons were made, where is the result of 10-fold dilu ons compared to ID NOW and other assays, what is the limited of detec on of Lucira?Table 1 (formerly Table S1) has been moved into the body of the manuscript to show this data and the methods describe the serial dilu ons.

LAMP might have cross-contamina on issues, how did authors address that; are those FP due to crosscontamina on?
Lucira tes ng was administered in clinical se ngs in the closed portable instrument, and therefore crosscontamina on from other specimens or from prior amplifica on would be unlikely.This sentence has been added to the discussion on lines 155-159.
What are the disadvantages of LAMP-based Lucira assay, will the me-to-result get longer for low-ter virus samples?Time to results for a specimen with a high viral load can be as li le as 10 min while nega ve or posi ve results from specimens with low viral loads take up to 30 min.Given the transport me to labs to perform NAAT tes ng, quick tes ng even at (~30 min) is a significant improvement.Other benefits of Lucira and limita ons have been added to the discussion.

What about the cost of Lucira assay compared to other rapid NAAT assays in terms of cost per sample, is it significantly cheaper?
Cost analyses were not possible given that Lucira kits, NAAT reagents, and consumables for clinical tes ng were provided by provincial and federal ini a ves to support SARS-CoV-2 tes ng.However, this was added as a study limita on.See lines 189-191.

Reviewer 2 Comment 1:
• The authors should provide an explanation in Table 1 for the absence of determined sensitivity in the perioperative diagnostic test and possibly include the PPV as well.Sensitivity and PPV was unable to be calculated to do an absence of false negative tests and true positive tests in the pre-operative setting.This was added to the Table 2 footnote (formerly Table 1).
• I am unsure about the meaning of "perioperative" as used by the authors.Using the term "preoperative screening" might be more appropriate.
Perioperative was changed to pre-operative throughout the manuscript.
• The sensitivity of the diagnostic tests in the emergency department was not sufficiently high.This aspect requires constructive criticism and a thorough examination of its limitations.We have added a sentence in the discussion to justify the rationale that the sensitivity of Lucira was deemed acceptable.See lines 132-134: "While the sensitivity and specificity were slightly lower than commercial NAATs used in clinical laboratories (Table 1) [12][13][14][15], the performance of Lucira was found to be acceptable to expedite SARS-CoV-2 results given it was equivalent to a LDT being used for diagnostic testing (Table 1)." • The authors asserted that the majority of patients from both the pre-operative screening and emergency conditions were asymptomatic (lacking respiratory symptoms).If the authors concur, they should provide the rationale behind this observation and the pretest likelihood ratio and engage in further discussion on this matter.Data on patients presenting to emergency departments (symptomatic or asymptomatic) was not available, and this has been added as a study limitation.Investigating differences between symptomatic and asymptomatic individuals was added to the study future direction.See lines 186-187.

Table 1 should include the PPV and NPV values.
PPV and NPV were added to Table 2 (formerly Table 1), when possible.

Comment 2:
The authors should explain the sample size calculation for all settings, but not for only one of perioperative, ED, and community cases.Additionally, to improve clarity, all settings groups in Table 1 should be displayed in the first row of the table.Sample sizes for each setting were dictated by the number of available participants over the timeframe of the quality initiative.

Comment 3:
Both the abstract and manuscript should be written concisely using well-refined English to enhance overall readability and quality.English and grammar has been reviewed throughout the manuscript.

Comment 4:
The authors should offer clear definitions of NAAT (Nucleic Acid Amplification Test) and NAAT-RDT (Nucleic Acid Amplification Test -Rapid Diagnostic Test) and consistently use these defined terms throughout the entire manuscript.These terms were defined in the background section (lines 67-72) and have been used consistently thereafter.

Comment 5:
The authors should provide a comprehensive definition of asymptomatic close contacts of COVID-19 cases and elucidate the reasons why evaluating these patients is essential for the study's objectives.Sentences were added to explain that healthcare workers with close contacts to positive SARS-CoV-2 cases were instructed to remain home for seven days following exposure.Asymptomatic testing was implemented to help these individuals return to work if test results were negative, reducing concerns of subsequent transmission.

Comment 6:
The authors should provide information on the Cycle threshold (Ct) value of NAAT in true positive (TP) and true negative (TN) results of NAAT-RDT for better understanding and analysis.These values were provided in the discussion with possible explanations.

Comment 7:
Regarding the claim of real-world testing, it is essential for the authors to specify the total number of both symptomatic and asymptomatic patients who underwent NAAT-RDT testing to provide a clearer context for the study's applicability.All patients in the pre-operative and close contact/HCW groups were asymptomatic as described in the methods.Based on the nature of this quality improvement project, it is not possible to determine symptom status of the ED patients which was described but has now been re-emphasized as a limitation in the discussion.

Comment 8:
The implementation of this study title seems to focus on asymptomatic individuals rather than asymptomatic individuals suspected to have COVID-19.However, it would be beneficial if the authors could present more data regarding the sensitivity and specificity in symptomatic COVID-19 cases.These data should be cited in the introduction and appropriately referenced.Comparisons between the performance of Lucira in symptomatic and asymptomatic individuals was found in the discussion, but we have better emphasized this topic in comparison to the only other study who reported Lucira test performance data (lines 135-139): "A single other study by Zahavi et al. [4] looked at the real-world performance of Lucira compared to NAATs [4], and the reported an overall sensitivity of 91.1% (95% CI: 83.2 to 96.1%) and specificity of 100.0 (95% CI: 96.4% to 100.0%).In their subset of symptomatic individuals, sensitivity was 93.1% (95% CI: 84.5% to 97.7%).Both sets of data were nearly identical to those found in our quality initiative which was primarily focused on asymptomatic testing."We have added to the discussion many possible factors that could explain the two discrepant results.

Comment 10:
In Line 134, the authors suggest that false negative Lucira results may be due to diagnostic failures resulting from sequence mismatches between the Lucira target and circulating SARS-CoV-2.However, to provide a more comprehensive understanding, the introduction should include additional details about the basis and principles of the Lucira test, particularly regarding the gene detection part before discussing the possibility of needing to resort to sequencing.The background has been modified to add more about the principle of Lucira, and the discussion has revised the content for sequencing and added a reference to support the situations where sequencing should be considered.

Comment 11:
In Line 137, the authors acknowledge that false positive (FP) and false negative (FN) results can occur with any diagnostic test.It is important to note that while these occurrences are possible for any test, for nucleic acid amplification tests (NAATs) in general, higher sensitivity and specificity are usually expected, often nearing 100%.This has been elaborated on in the discussion.

Comment 12:
The authors should expand on the limitations of the study to provide a more comprehensive assessment of potential constraints or factors that might affect the interpretation of the results.Addressing these limitations will contribute to a more well-rounded discussion of the study's findings.Additional limitations and future directions have been added to the discussion.
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The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.The author should be asked to provide reference values for SARS-CoV-2 concentrations in Table 1 that are correlated with the Lucira Ct values, especially when using a standard virus for measurement.This would enhance the clarity of the data.
Comment 2: It's recommended to include the sentence "majority of asymptomatic settings in WHO screening in pre-operative and community" somewhere in the text for context.
Comment 3: In Table 2, there seems to be confusion when calculating TP, TN, FP, FN in each setting, leading to incorrect numbers.The authors should refer to the 2x2 table to ensure accurate numbers and adhere to the STARD guideline for reporting.Careful review of the results is necessary.
Comment 4 Line 107: True positive and true negative results were defined by concordant results between Lucira and one of the three commercial NAATs.To ensure clarity and reliability, it's advisable to choose a single reference standard PCR test and provide detailed information about this test, including the cutoff value used.This will establish a clear benchmark for comparison in the study and enhance the validity of the results.Comment 5 Please provide a flowchart of participant enrollment and characteristics according to the STARD guideline to enhance clarity.

Comment 6
In the discussion section, it would be beneficial to present the main results or conclusions before delving into details.

Comment 7
Reduce the use of the term "Lucira" and consider using terms like "NAAT-RDT" or "POC testing" instead for variety and clarity.
Comment 8 Line 132: While the sensitivity and specificity were slightly lower than commercial NAATs used in clinical laboratories (Table 1) Certainly, emphasizing the term "standard NAATs" over "commercial NAATs" in the discussion and throughout the paper would provide greater clarity and alignment with established testing practices.
Comment 9 Line 146: commercial NAATs are known to be more sensitive than LDTs and NAAT-RDTs using isothermal amplification technologies [3,[12][13][14][15][16][17], therefore failure of Lucira to detect SARS-CoV-2 in specimens with low viral loads was not surprising.I suggest the authored remove and replace with interpretation comparing with the standard reference test.Comment 10 Line 150: On the other hand, the other two false negative Lucira results had paired specimens with low Ct values of 18.4 and 18.9 when tested on a commercial NAAT, suggesting the specimens had high viral loads seen during the infectious period of SARS-CoV-2.There are multiple possibilities that could explain these discrepant results.To ensure Lucira was able to detect specimens with high viral loads, 5 specimens with Xpert Ct values spanning 16.5 to 17.5 were tested and were all detected within 10 minutes (data not shown).I should in this point the author should carefully discuss and provide the result in supplementary.
Comment 11 Line 155: Please remove "Second, Lucira testing was administered at the point-of-care in with the closed portable single-use instrument, and therefore cross-contamination between specimens or from prior testing in the same location would be unlikely; however, contamination could be possible in a clinical laboratory with the NAAT used as the comparator (i.e., Xpert in these two cases)."Your suggestion to carefully interpret the findings and review potential sources of bias, including pre-analytical, analytical, and post-analytical factors, is valid.This thorough examination of errors and biases can help to better understand the reliability of the study and the selection of the standard test.It's important to consider and address these factors in the interpretation of the results for a more comprehensive and accurate assessment.
Comment 12 Line 164: It is possible that both patients with discrepant result were in an early acute stage of illness detectable in the laboratory by a commercial NAAT, but SARS-CoV-2 was not detected with Lucira 24h prior as the viral load was below the level of detection.Your point regarding the need for a detailed comparison of methods and processing between Lucira and standard NAATs is valid.If there are differences in specimen collection, handling, or processing, these could indeed contribute to false negatives or other discrepancies in results.Addressing these differences and their potential impact on the study's findings would enhance the overall reliability and interpretation of the results.It's important for the authors to provide a comprehensive understanding of the testing procedures to assess potential sources of error and bias.
Comment 13 Education and training focused on the importance of proper specimen collection, timing of collection, and testing could help avoid possible false negative results and SARS-CoV-2 transmissions The authors should declare this training and method in the early of manuscript.
Comment 14 Line 187: Another main limitation of this quality initiative is the absence of data available to assess the cost-benefits and impact of Lucira implementation in the testing settings.Cost analyses were not possible given Lucira kits and NAAT reagents and consumables for clinical testing were provided by provincial and federal initiatives to support SARS-CoV-2 testing.I think this issue did not relate to this topic.

Comment 15:
The language in limitation part should be revised.

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Response to Reviewer Comments
Comment 1: The author should be asked to provide reference values for SARS-CoV-2 concentrations in Table 1 that are correlated with the Lucira Ct values, especially when using a standard virus for measurement.This would enhance the clarity of the data.Please see the first column of Table 1 for reference values of SARS-CoV-2 concentrations in copies/mL.

Comment 2:
It's recommended to include the sentence "majority of asymptomatic settings in WHO screening in pre-operative and community" somewhere in the text for context.The authors are not familiar with WHO screening recommendations/criteria in his context.We would request the reviewer clarify this or provide a resource for us to consult.

Comment 3:
In Table 2, there seems to be confusion when calculating TP, TN, FP, FN in each setting, leading to incorrect numbers.The authors should refer to the 2x2 table to ensure accurate numbers and adhere to the STARD guideline for reporting.Careful review of the results is necessary.
The results displayed in Table 2 have been reviewed.No errors were found.

Comment 4
Line 107: True positive and true negative results were defined by concordant results between Lucira and one of the three commercial NAATs.To ensure clarity and reliability, it's advisable to choose a single reference standard PCR test and provide detailed information about this test, including the cutoff value used.This will establish a clear benchmark for comparison in the study and enhance the validity of the results.In order to maximize laboratory efficiency while processing high sample volumes during the SARS-CoV-2 pandemic, several standard NAATs were used in our lab at the time of data collection.As outlined in Table 1, results were comparable amongst NAATs used.

Comment 5
Please provide a flowchart of participant enrollment and characteristics according to the STARD guideline to enhance clarity.This quality initiative did not collect any data on participant characteristics.

Comment 6
In the discussion section, it would be beneficial to present the main results or conclusions before delving into details.Please see paragraph one of the discussion section for main results and conclusions (lines 130-137).

Comment 7
Reduce the use of the term "Lucira" and consider using terms like "NAAT-RDT" or "POC testing" instead for variety and clarity.This edit has been made in the manuscript.

Comment 8
Line 132: While the sensitivity and specificity were slightly lower than commercial NAATs used in clinical laboratories (Table 1) Certainly, emphasizing the term "standard NAATs" over "commercial NAATs" in the discussion and throughout the paper would provide greater clarity and alignment with established testing practices.This edit has been made in the manuscript.The purpose of performing this additional testing was to exclude template inhibition as a possible reason for false negative tests.This is described in the section where false negatives are discussed.
Comment 11 Line 155: Please remove "Second, Lucira testing was administered at the point-of-care in with the closed portable single-use instrument, and therefore cross-contamination between specimens or from prior testing in the same location would be unlikely; however, contamination could be possible in a clinical laboratory with the NAAT used as the comparator (i.e., Xpert in these two cases)."Your suggestion to carefully interpret the findings and review potential sources of bias, including pre-analytical, analytical, and post-analytical factors, is valid.This thorough examination of errors and biases can help to better understand the reliability of the study and the selection of the standard test.It's important to consider and address these factors in the interpretation of the results for a more comprehensive and accurate assessment.
We have reworded this section to recognize the possibility of false positive or false negative results occurring from either the NAAT-RDT or the standard NAATs in the laboratory.

Comment 12
Line 164: It is possible that both patients with discrepant result were in an early acute stage of illness detectable in the laboratory by a commercial NAAT, but SARS-CoV-2 was not detected with Lucira 24h prior as the viral load was below the level of detection.Your point regarding the need for a detailed comparison of methods and processing between Lucira and standard NAATs is valid.If there are differences in specimen collection, handling, or processing, these could indeed contribute to false negatives or other discrepancies in results.Addressing these differences and their potential impact on the study's findings would enhance the overall reliability and interpretation of the results.It's important for the authors to provide a comprehensive understanding of the testing procedures to assess potential sources of error and bias.The potential of improper collection, handling, or processing was mentioned in lines 176-178.

Comment 13
Education and training focused on the importance of proper specimen collection, timing of collection, and testing could help avoid possible false negative results and SARS-CoV-2 transmissions The authors should declare this training and method in the early of manuscript.This has been added to the study design section: see lines 112-113.

Comment 14
Line 187: Another main limitation of this quality initiative is the absence of data available to assess the cost-benefits and impact of Lucira implementation in the testing settings.Cost analyses were not possible given Lucira kits and NAAT reagents and consumables for clinical testing were provided by provincial and federal initiatives to support SARS-CoV-2 testing.I think this issue did not relate to this topic.Cost remains an important practical consideration when evaluating any laboratory test.

Comment 15:
The language in limitation part should be revised.The language in this section has been reviewed.
Comment 9: In Line 130, the authors mentioned that the other two false negative results from the Lucira NAAT-RDT had paired specimens with low Ct values of 18.4 and 18.9, indicating high viral loads during the infectious period of SARS-CoV-2.The authors should elaborate on why negative NAAT-RDT results can occur during a period of high viral load to provide a more comprehensive understanding of the test's performance under such circumstances.
Comments for the Author): my comments and questions have been addressed.Reviewer #2 (Comments for the Author): Comment 1: • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred NAATs are known to be more sensitive than LDTs and NAAT-RDTs using isothermal amplification technologies [3, 12-17], therefore failure of Lucira to detect SARS-CoV-2 in specimens with low viral loads was not surprising.I suggest the authored remove and replace with interpretation comparing with the standard reference test.This line has been edited.Comment 10 Line 150: On the other hand, the other two false negative Lucira results had paired specimens with low Ct values of 18.4 and 18.9 when tested on a commercial NAAT, suggesting the specimens had high viral loads seen during the infectious period of SARS-CoV-2.There are multiple possibilities that could explain these discrepant results.To ensure Lucira was able to detect specimens with high viral loads, 5 specimens with Xpert Ct values spanning 16.5 to 17.5 were tested and were all detected within 10 minutes (data not shown).I should in this point the author should carefully discuss and provide the result in supplementary.