New Paper (Diagnostic): A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

[jessegmeyerlab]

Title: A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

Please paste a link to the paper or a citation here:

Link: https://www.nature.com/articles/s41598-021-95799-6

What is the paper's Manubot-style citation?

Citation: [@url:https://www.nature.com/articles/s41598-021-95799-6]

Please list some keywords (3-10) that help identify the relevance of this paper to COVID-19

• RT-LAMP
• nucleic acid test
• molecular test
• point-of-care

Please note the publication / review status

• Pre-print
• New Peer-Reviewed Paper
• Peer-Reviewed Paper Pre-2020

Which areas of expertise are particularly relevant to the paper?

• virology
• epidemiology
• biostatistics
• immunology
• pharmacology
• analytical chemistry

Questions to answer about each paper:

Please provide 1-2 sentences introducing the study and its main findings

RT-PCR remains the gold standard for detection of SARS-CoV-2 RNA from infected patients, but the traditional method requires special equipment and reagents, especially a thermocycler. This study shows that isothermal RT- Loop-mediated isothermal amplification (LAMP) is effective for detection of SARS-CoV-2 with excellent specificity and sensitivity, and that this method can be applied to unprocessed saliva samples.

Study question(s) being investigated:

What type of testing scenario is being considered?

This test aims to bring the sensitivity of nucleic acid detection to the point of care or home testing setting. It could be applied for screening, diagnostics, or as a definitive test for people who are positive based on lateral flow tests.

Study population:

What is the model system (e.g., human study, animal model, cell line study)?

The was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples.

What is the sample size?

177

What is the "pre-test" probability of disease in the study population (i.e., what is the anticipated prevalence of the disease?)

This new test was not applied to discover SARS-CoV-2 in prospective cohorts, but rather it was validated against samples already collected. 126/177 of the human nasopharyngeal RNA samples were positive according to RT-PCR.

For human studies, the following are related to the pre-test probability:

What countries/regions are considered?

This seems not applicable, but the validation set of samples appear to be from Portugal.

What is the age range, gender, other relevant characteristics?

Does not appear to be mentioned.

What is the setting of the study (e.g., random sample of school children, retirement communities, etc.)?

Not mentioned.

What other specific inclusion-exclusion criteria are considered?

Not mentioned.

Reference test:

What reference test is considered as a "gold standard" comparator for the test under investigation?

RT-PCR

Test assignment:

How are the new and reference tests assigned?

The same samples that were positive by RT-PCR were tested by RT-LAMP.

Are there any other relevant details about the study design?

The authors do break down the sensitivity of their test according to the cycle threshold (Ct) value from the RT-PCR of the same samples and show that RT-LAMP performs at 100% sensitivity for samples with a Ct of 32 or less (Figure 2). The performance is worse when considering any positive sample (including Ct values of 32-40).

Test conduct:

How were tests performed?

Describe technical details of assays used, when measurements were taken and by whom, etc. for both the new and standard tests.

RT-LAMP: They used various combinations of reagents, but an example is using WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) with a set of six primers developed previously by Zhang et al (https://doi.org/10.1101/2020.02.26.20028373). To determine assay sensitivity they used serial tenfold dilutions of in vitro transcribed N-gene RNA standard (IVT RNA), starting from 10^5 copies down to 10 copies. The readout is the color of their dye changing proportional to the DNA product over 30 minutes. They then applied this test to the clinical nasopharyngeal samples.

Test Assessment

Describe how individuals are classified as positive or negative, e.g. if a threshold is used.

If the solution changes from red to yellow after 30 minute incubation at 65 degrees C they are positive.

Is there evidence that the test is precise/reproducible when repeated more than once?

Are measurements complete?

If the test is positive it is unlikely a false positive but the test is not as sensitive as RT-PCR and a negative test could be a false negative.

Results summary:

What are the estimated sensitivity, specificity, positive predictive value (PPV), and negative predicted value (NPV)?

"For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%."
"the direct RT-LAMP assay had a sensitivity of 85% (CI 70–93%) for saliva samples with matched NP swabs with Ct ≤ 28 (Figure 4)"

These values are obtained by comparison with RT-PCR results.

What are the confidence bounds around these intervals?

The confidence bounds are only given for the direct RT-LAMP.

Interpretation of results for study population:

How good is the test at ruling in or ruling out a disease based on the post-test probabilities?

Post-test probabilities were not given.

Are there identified side affects of the test?

None.

Is patient adherence to the test likely to be an issue?

No.

Extrapolation of conclusions to other groups of individuals

This test should be widely applicable like RT-PCR. It may enable under resourced areas to get better testing.

How well is the test likely to work in populations with different pretest odds?

For example, if the prevalence is lower, then the PPV will also be lower, but the NPV will be higher.

How costly is the test?

Estimated at 2 euros per test.

How difficult is it to perform the test in different settings?

It should be easier to perform remote from a lab environment.

Could the test be combined with other existing tests?

This test is meant to replace RT-PCR.

Summary of reliability

The main strength of this test over RT-PCR is that it can be done isothermally, but the main drawback is about 10-fold less sensitivity than RT-PCR.

Progress

Check off the components as they are completed. If the component is not applicable, check the box as well.

• 1-2 sentences introducing the study and its main findings
• Describe testing scenario
• Describe model system
• Sample size
• Describe prevalence of disease
• Describe countries/regions are considered
• Describe age range, gender, other relevant characteristics
• Describe setting of the study
• Describe other specific inclusion-exclusion criteria
• Describe "gold standard"
• Describe how the new and reference tests assigned
• Describe other relevant details about the study design
• Describe how the tests were performed
• Describe how individuals are classified as positive or negative
• Describe if test is precise/reproducible
• Describe whether measurements are complete
• What are the estimated sensitivity, specificity, positive predictive value (PPV), and negative predicted value (NPV)?
• What are the confidence bounds around these intervals?
• Describe post-test probabilities
• Describe side affects of the test
• Describe patient adherence
• Describe how it will extrapolate
• How costly is the test?
• How difficult is it to perform the test in different settings?
• Could the test be combined with other existing tests?
• Summary of reliability