From b3970515ab2584110e3b7a1d63672a5e169cb26d Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Wed, 29 Dec 2021 08:57:02 -0800 Subject: [PATCH 01/24] text structure and minor edits break up long intro paragraphs, shortened wording, and added information including citation --- content/10.diagnostics.md | 18 +++++++++++------- 1 file changed, 11 insertions(+), 7 deletions(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index aa0b6d146..eae21f121 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -15,13 +15,17 @@ For instance, test-trace-isolate procedures were an early cornerstone of many na The genetic sequence of the virus was first released by Chinese officials on January 10, 2020, and the first test to detect the virus was released about 13 days later [@doi:10.2807/1560-7917.ES.2020.25.3.2000045]. This information is important to the development of diagnostic approaches using a variety of approaches. There are two main classes of diagnostic tests: molecular tests, which can diagnose an active infection by identifying the presence of SARS-CoV-2, and serological tests, which can assess whether an individual was infected in the past via the presence or absence of antibodies against SARS-CoV-2. -Molecular tests are essential for identifying individuals for treatment and alerting their contacts to quarantine and be alert for possible symptoms. -Here, a patient sample is evaluated to determine the presence or absence of a viral target. -These techniques depend on knowledge of the viral genomic sequence for the development of targeted primers. -On the other hand, serological tests are useful for collecting population-level information for epidemiological analysis, as they can be used to estimate the extent of the infection in a given area. -Thus, they may be useful in efforts to better understand the percent of cases that manifest as severe versus mild and for guiding public health and economic decisions regarding resource allocation and counter-disease measures. -These tests typically detect the presence of antibodies in blood plasma samples. -In such enzyme-linked immunosorbent assay (ELISA) approaches, the detection of the antibodies depends on knowledge of a specific antibody-antigen interaction. + +Molecular tests are essential to identify sick individuals who may need treatment and whose contacts should quarantine in anticipation of COVID-19 symptoms. +Molecular tests determine the presence or absence of a viral target in a patient sample, most often from a nose or throat swab. +Molecular tests detect either viral RNA via polymerase chain reaction (PCR) methods or viral protein via lateral flow test (LFT). +Thus, these techniques depend on knowledge of the viral genomic sequence for the development of targeted primers. + +Serological tests are useful for collecting population-level information for epidemiological analysis, as they can be used to estimate the extent of the infection in a given area. +Thus, serological tests may be useful to better understand the percent of cases that manifest as severe versus mild and for guiding public health and economic decisions regarding resource allocation and counter-disease measures. +Serological tests detect the presence of antibodies in blood plasma samples. +In such enzyme-linked immunosorbent assay (ELISA) approaches, the detection of the antibodies depends on knowledge of a specific antibody-antigen interaction. +Because baccines are based on the viral spike protein, to distinguish past infection from vaccination, serology tests look for antibodies that bind the nucleocapsid protein of the SARS-CoV-2 virus[@doi:10.1093/infdis/jiaa273]. As the pandemic has evolved throughout 2020 and 2021, a variety of technological implementations have emerged within these two categories. From 31d53f43c1362990f5a300b6e4f6d971f52b6caf Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Wed, 29 Dec 2021 09:15:33 -0800 Subject: [PATCH 02/24] Update 10.diagnostics.md add subsection for sequencing and LFTs --- content/10.diagnostics.md | 15 ++++++++++++--- 1 file changed, 12 insertions(+), 3 deletions(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index eae21f121..002e91caf 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -44,12 +44,21 @@ Policy discussions have also reviewed both the critical importance of diagnostic Molecular tests are used to identify distinct genomic subsequences of a viral molecule in a sample and thus to diagnose an active viral infection. An important first step is identifying which biospecimens are likely to contain the virus in infected individuals and then acquiring these samples from the patient(s) to be tested. Common sampling sources for molecular tests include nasopharyngeal cavity samples, such as throat washes, throat swabs, and saliva [@doi:10/ggp4qx], and stool samples [@doi:10.1002/jmv.25742]. -Once a sample from an appropriate source is acquired from a patient, molecular tests follow a number of different steps, described below, to analyze a sample and identify whether evidence of SARS-CoV-2 is present. -When testing for RNA viruses like SARS-CoV-2, the first step involves pre-processing in order to create complimentary DNA (cDNA) from the RNA sample using reverse transcription (RT). +Once a sample from an appropriate source is acquired from a patient, molecular tests follow a number of different steps, described below, to analyze a sample and identify whether evidence of SARS-CoV-2 is present. There are two important categories of molecular tests: PCR-based tests and LFT-based tests. + +LFT-based tests are beneficial because they can detect current infection within 30 minutes and because they can be performed without specialized equipment at low cost. +LFT methods rely on detection of the viral protein with an antibody. +LFTs are routinely used to detect a variety of diseases and even drugs of abuse in urine (citations). + +When testing for RNA viruses like SARS-CoV-2 with PCR-based tests, the first step involves pre-processing in order to create complimentary DNA (cDNA) from the RNA sample using reverse transcription (RT). The second step involves the amplification of a region of interest in the cDNA using successive cycles of heating and cooling. Depending on the application, different polymerase chain reaction (PCR) procedures exist, which are described below. + + + +#### SEQUENCING Some tests use the results of the PCR itself to determine whether the pathogen is present, but in other cases, it may be necessary to sequence the amplified DNA. -For sequencing, an additional pre-processing step, library preparation, therefore must be undertaken. +Sequencing requires an additional sample pre-processing step called library preparation. Library preparation is the process of preparing the sample for sequencing, typically by fragmenting the sequences and adding adapters [@doi:10.1016/j.biotechadv.2020.107537]. In some cases, library preparation can involve other modifications of the sample, such as adding barcodes to identify a particular sample within the sequence data. Barcoding can therefore be used to pool samples from multiple sources. From 67eddfc23f75d2e389b8ed0b04d3046cae1327ae Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Wed, 29 Dec 2021 12:37:31 -0800 Subject: [PATCH 03/24] Update 10.diagnostics.md added section on RT LAMP with one review cited and one recent study cited. Also added a section on lateral flow tests as to-do. Will need to add more examples of both. Also changed some organization and formatting, for example moved sequencing to its own section, which was previously part of the intro to molecular tests section. --- content/10.diagnostics.md | 52 ++++++++++++++++++++++++++++++--------- 1 file changed, 41 insertions(+), 11 deletions(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 002e91caf..1b4d96050 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -44,19 +44,14 @@ Policy discussions have also reviewed both the critical importance of diagnostic Molecular tests are used to identify distinct genomic subsequences of a viral molecule in a sample and thus to diagnose an active viral infection. An important first step is identifying which biospecimens are likely to contain the virus in infected individuals and then acquiring these samples from the patient(s) to be tested. Common sampling sources for molecular tests include nasopharyngeal cavity samples, such as throat washes, throat swabs, and saliva [@doi:10/ggp4qx], and stool samples [@doi:10.1002/jmv.25742]. -Once a sample from an appropriate source is acquired from a patient, molecular tests follow a number of different steps, described below, to analyze a sample and identify whether evidence of SARS-CoV-2 is present. There are two important categories of molecular tests: PCR-based tests and LFT-based tests. - -LFT-based tests are beneficial because they can detect current infection within 30 minutes and because they can be performed without specialized equipment at low cost. -LFT methods rely on detection of the viral protein with an antibody. -LFTs are routinely used to detect a variety of diseases and even drugs of abuse in urine (citations). +Once a sample from an appropriate source is acquired from a patient, molecular tests follow a number of different steps, described below, to analyze a sample and identify whether evidence of SARS-CoV-2 is present. There are two important categories of molecular tests: (1) nucleic acid tests based on PCR and (2) protein tests based on LFT. +#### Nucleic acid tests When testing for RNA viruses like SARS-CoV-2 with PCR-based tests, the first step involves pre-processing in order to create complimentary DNA (cDNA) from the RNA sample using reverse transcription (RT). The second step involves the amplification of a region of interest in the cDNA using successive cycles of heating and cooling. Depending on the application, different polymerase chain reaction (PCR) procedures exist, which are described below. - - -#### SEQUENCING +##### SEQUENCING Some tests use the results of the PCR itself to determine whether the pathogen is present, but in other cases, it may be necessary to sequence the amplified DNA. Sequencing requires an additional sample pre-processing step called library preparation. Library preparation is the process of preparing the sample for sequencing, typically by fragmenting the sequences and adding adapters [@doi:10.1016/j.biotechadv.2020.107537]. @@ -67,7 +62,7 @@ Sequential pattern matching is then used to identify unique subsequences of the Therefore, tests that utilize sequencing require a number of additional molecular and analytical steps relative to tests that use PCR alone. -#### RT-PCR +##### RT-PCR Reverse Transcription Polymerase Chain Reaction (RT-PCR) tests determine whether a target is present by amplifying a region of interest of cDNA [@doi:10.1042/BIO20200034]. @@ -78,7 +73,7 @@ Specifically this test utilizes two probes against RdRP, one of which is specifi Importantly, this assay was not found to return false positive results. -#### qPCR +##### qPCR In this manuscript we refer to quantitative real-time PCR as qPCR, following the recommendations of the MIQE guidelines [@doi:10.1373/clinchem.2008.112797]. In contrast to RT-PCR, qPCR uses fluorescent dyes that bind to the amplified DNA, thereby allowing a real time assessment of the amplification procedure [@doi:10.1042/BIO20200034]. @@ -123,7 +118,34 @@ This evidence further indicates that the lower limit of detection made possible Overall, these studies suggest that ddPCR is a promising tool for overcoming the problem of false-negative SARS-CoV-2 testing. -#### Pooled and Automated PCR Testing + +##### RT-LAMP +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. +Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) is an alternative to traditional PCR that does not require a thermal cycler. +RT-LAMP pilot studies for detecion of SARS-CoV-2 were recently reviewed and meta-analyzed [@PMID:34086539]. +In the meta analysis of all 2,112 samples, the cumulative sensitivity of RT-LAMP was 95.5% (CI 97.5% = 90.8-97.9%) and the cumulative specificity was 99.5% (CI 97.5% = 97.7-99.9%). +The low cost, sensitity/specificity, and quick readout of RT-LAMP makes this an attractive alternative to RT-PCR. +Alternative strategies like RT-LAMP are needed to bring widespread testing to rural or under resourced areas. + + +One recent study showed that RT-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 [@url:https://www.nature.com/articles/s41598-021-95799-6]. +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. +The new method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. +The authors 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. +The performance is worse when considering any positive sample (including Ct values of 32-40). +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 [@URL: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. +For viral loads above 100 copies of genomic RNA, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1% from purified RNA. +The direct assay of saliva by RT-LAMP had a sensitivity of 85% (CI 70-93%). +Sensitivity and specificity metrics were obtained by comparison with results from RT-PCR. +The estimated cost per test is about 2 euros. +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. + + +##### Pooled and Automated PCR Testing Due to limited supplies and the need for more tests, several labs have found ways to pool or otherwise strategically design tests to increase throughput. The first such result came from Yelin et al. [@doi:10.1101/2020.03.26.20039438], who found they could pool up to 32 samples in a single qPCR run. @@ -134,6 +156,7 @@ Technology based on CRISPR (clustered regularly interspaced short palindromic re ##### CRISPR-based Detection +Technology based on CRISPR (clustered regularly interspaced short palindromic repeats) [@DOI:https://doi.org/10.1038/s41467-018-04252-2] has also been instrumental in scaling up testing protocols. Two CRISPR-associated nucleases, Cas12 and Cas13, have been used for nucleic acid detection. Multiple assays exploiting these nucleases have emerged as potential diagnostic tools for the rapid detection of SARS-CoV-2 genetic material and therefore SARS-CoV-2 infection. The SHERLOCK method (Specific High-sensitivity Enzymatic Reporter unLOCKing) from Sherlock Biosciences relies on Cas13a to discriminate between inputs that differ by a single nucleotide at very low concentrations [@doi:10.1126/science.aam9321]. @@ -175,6 +198,13 @@ Both techniques were equally sensitive in detecting SARS-CoV-2. Lateral flow strips showed a 100% correlation to the high-throughput DETECTR assay. Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other human coronaviruses. + + + + #### Limitations of Molecular Tests Tests that identify SARS-CoV-2 using nucleic-acid-based technologies will identify only individuals with current infections and are not appropriate for identifying individuals who have recovered from a previous infection. From 98229e053d1ef968848a3c3a6d1c619db5add9ab Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Thu, 30 Dec 2021 06:52:58 -0800 Subject: [PATCH 04/24] Update 10.diagnostics.md added minimum of LFT section under molecular tests, minor edits elsewhere --- content/10.diagnostics.md | 68 ++++++++++++++++++++++----------------- 1 file changed, 38 insertions(+), 30 deletions(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 1b4d96050..c2303dc30 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -13,22 +13,21 @@ For instance, test-trace-isolate procedures were an early cornerstone of many na The genetic sequence of the virus was first released by Chinese officials on January 10, 2020, and the first test to detect the virus was released about 13 days later [@doi:10.2807/1560-7917.ES.2020.25.3.2000045]. -This information is important to the development of diagnostic approaches using a variety of approaches. +This information is important to the development of diagnostic approaches. There are two main classes of diagnostic tests: molecular tests, which can diagnose an active infection by identifying the presence of SARS-CoV-2, and serological tests, which can assess whether an individual was infected in the past via the presence or absence of antibodies against SARS-CoV-2. +As the pandemic has evolved throughout 2020 and 2021, a variety of tests have emerged within these two categories. -Molecular tests are essential to identify sick individuals who may need treatment and whose contacts should quarantine in anticipation of COVID-19 symptoms. -Molecular tests determine the presence or absence of a viral target in a patient sample, most often from a nose or throat swab. -Molecular tests detect either viral RNA via polymerase chain reaction (PCR) methods or viral protein via lateral flow test (LFT). -Thus, these techniques depend on knowledge of the viral genomic sequence for the development of targeted primers. +Molecular tests detect either viral RNA or protein in a patient sample. +Molecular tests are essential to identify infected individuals who may need treatment and whose contacts should quarantine. +Tests for viral RNA are done by reverse transcription (RT) of viral RNA to DNA followed by DNA amplification, usually by the polymerase chain reaction (PCR) [@DOI:10.1038/jid.2013.1]. +Tests for viral proteins are most often done using detection by an antibody pair in lateral flow tests (LFTs) [@URL:https://doi.org/10.1007/s00216-010-3661-4]. +Molecular tests require the viral genome sequence to develop DNA primers for viral RNA detection, or to express a viral protein for use as an antigen in antibody production. +Serological tests detect the presence of antibodies in blood plasma samples using enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA) [@DOI:10.3389/fmolb.2021.682405]. +Because vaccines are based on the viral spike protein, to distinguish past infection from vaccination, serology tests look for antibodies that bind the nucleocapsid protein of the SARS-CoV-2 virus[@doi:10.1093/infdis/jiaa273]. Serological tests are useful for collecting population-level information for epidemiological analysis, as they can be used to estimate the extent of the infection in a given area. Thus, serological tests may be useful to better understand the percent of cases that manifest as severe versus mild and for guiding public health and economic decisions regarding resource allocation and counter-disease measures. -Serological tests detect the presence of antibodies in blood plasma samples. -In such enzyme-linked immunosorbent assay (ELISA) approaches, the detection of the antibodies depends on knowledge of a specific antibody-antigen interaction. -Because baccines are based on the viral spike protein, to distinguish past infection from vaccination, serology tests look for antibodies that bind the nucleocapsid protein of the SARS-CoV-2 virus[@doi:10.1093/infdis/jiaa273]. -As the pandemic has evolved throughout 2020 and 2021, a variety of technological implementations have emerged within these two categories. - @@ -44,12 +43,13 @@ Policy discussions have also reviewed both the critical importance of diagnostic Molecular tests are used to identify distinct genomic subsequences of a viral molecule in a sample and thus to diagnose an active viral infection. An important first step is identifying which biospecimens are likely to contain the virus in infected individuals and then acquiring these samples from the patient(s) to be tested. Common sampling sources for molecular tests include nasopharyngeal cavity samples, such as throat washes, throat swabs, and saliva [@doi:10/ggp4qx], and stool samples [@doi:10.1002/jmv.25742]. -Once a sample from an appropriate source is acquired from a patient, molecular tests follow a number of different steps, described below, to analyze a sample and identify whether evidence of SARS-CoV-2 is present. There are two important categories of molecular tests: (1) nucleic acid tests based on PCR and (2) protein tests based on LFT. +Once a sample is acquired from a patient, molecular tests detect SARS-CoV-2 based on the presence of either (1) viral nucleic acids and (2) viral protein. #### Nucleic acid tests -When testing for RNA viruses like SARS-CoV-2 with PCR-based tests, the first step involves pre-processing in order to create complimentary DNA (cDNA) from the RNA sample using reverse transcription (RT). +When testing for RNA from viruses like SARS-CoV-2, the first step involves pre-processing in order to create complimentary DNA (cDNA) from the RNA sample using reverse transcription (RT). The second step involves the amplification of a region of interest in the cDNA using successive cycles of heating and cooling. -Depending on the application, different polymerase chain reaction (PCR) procedures exist, which are described below. +Depending on the application, amplification is achieved using variations of the polymerase chain reaction (PCR). +Nucleic acid tests differ primarily in detection methods, which are detailed below. ##### SEQUENCING Some tests use the results of the PCR itself to determine whether the pathogen is present, but in other cases, it may be necessary to sequence the amplified DNA. @@ -60,6 +60,7 @@ Barcoding can therefore be used to pool samples from multiple sources. There are different reagents used for library preparation that are specific to identifying one or more target sections with PCR [@doi:10.1021/acsnano.0c02624]. Sequential pattern matching is then used to identify unique subsequences of the virus, and if sufficient subsequences are found, the test is considered positive. Therefore, tests that utilize sequencing require a number of additional molecular and analytical steps relative to tests that use PCR alone. +Sequencing has been an important strategy for discovery of SARS-CoV-2 variants (for example, see [@DOI:10.1101/2021.01.18.21249786]). ##### RT-PCR @@ -121,28 +122,28 @@ Overall, these studies suggest that ddPCR is a promising tool for overcoming the ##### RT-LAMP 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. -Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP) is an alternative to traditional PCR that does not require a thermal cycler. +Loop Mediated Isothermal Amplification (LAMP) is an alternative to PCR that does not require a thermal cycler [@DOI:10.1093/nar/28.12.e63]. +LAMP is combined with reverse transcription (RT-LAMP) to enable detection of RNA. RT-LAMP pilot studies for detecion of SARS-CoV-2 were recently reviewed and meta-analyzed [@PMID:34086539]. In the meta analysis of all 2,112 samples, the cumulative sensitivity of RT-LAMP was 95.5% (CI 97.5% = 90.8-97.9%) and the cumulative specificity was 99.5% (CI 97.5% = 97.7-99.9%). -The low cost, sensitity/specificity, and quick readout of RT-LAMP makes this an attractive alternative to RT-PCR. -Alternative strategies like RT-LAMP are needed to bring widespread testing to rural or under resourced areas. - +The low cost, excellent sensitity/specificity, and quick readout of RT-LAMP makes this an attractive alternative to RT-PCR. +Alternative strategies like RT-LAMP are needed to bring widespread testing away from the lab and into under resourced areas. One recent study showed that RT-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 [@url:https://www.nature.com/articles/s41598-021-95799-6]. 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. -The new method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. -The authors 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. -The performance is worse when considering any positive sample (including Ct values of 32-40). -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 [@URL: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. +This method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. +The authors break down the sensitivity of their test according to the cycle threshold (Ct) value from RT-PCR of the same samples; RT-LAMP performs at 100% sensitivity for samples with a Ct from RT-PCR of 32 or less. +The performance is worse when considering all RT-PCR positive samples (including those with Ct values between 32-40). +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 [@URL:https://doi.org/10.1101/2020.02.26.20028373]. +To determine assay sensitivity, serial tenfold dilutions of _in vitro_ transcribed N-gene RNA standard (IVT RNA) were tested using quantities from 10^5 copies down to 10 copies. +The assay readout is the color of dye changing from red to yellow due to binding to the DNA product over 30 minutes. +The RT-LAMP assay was then applied to clinical nasopharyngeal samples. For viral loads above 100 copies of genomic RNA, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1% from purified RNA. The direct assay of saliva by RT-LAMP had a sensitivity of 85% (CI 70-93%). Sensitivity and specificity metrics were obtained by comparison with results from RT-PCR. -The estimated cost per test is about 2 euros. -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. +The estimated cost per test is about 2 euros when RNA extraction is included. +The main strength of this test over RT-PCR is that it can be done isothermally, but the main drawback is that it is about 10-fold less sensitive than RT-PCR. ##### Pooled and Automated PCR Testing @@ -198,12 +199,19 @@ Both techniques were equally sensitive in detecting SARS-CoV-2. Lateral flow strips showed a 100% correlation to the high-throughput DETECTR assay. Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other human coronaviruses. - - - +Often this is done with an antibody sandwich format, where one antibody containing a dye is binds at one site on the antigen, and an immobilized antibody on the strip binds at another site, allowing accumulation of the dye and the characteristic formation of a positive test line on the strip [@URL:https://doi.org/10.1007/s00216-010-3661-4]. +The applications of LFTs are broad: they are routinely used for home pregnancy tests, disease detection, and even drugs of abuse detection in urine [@DOI:10.1007/s00216-008-2287-2]. +Current infection detection by LFTs is needed to enable the scale and speed of testing required to stop the spread of SARS-CoV-2. +Lateral flow tests were made available freely to citizens in the United Kingdom at least until December 2021 [@DOI:10.1136/bmj.n1760]. + +A recent review surveyed the performance of LFTs for detection of current SARS-CoV-2 infection [@PMID:34407759]. +This review covered 24 studies that included more than 26,000 total LFTs. +They found significant heterogeneity in test sensitivities ranging from 37.7% (95% CI 30.6-45.5) to 99.2% (95% CI 95.5-99.9). +The specificities of these tests were more homogeneous, spanning 92.4% (95% CI 87.5-95.5) to 100.0% (95% CI 99.7-100.0). + #### Limitations of Molecular Tests From 68f1b66af54376995745a527c653d3c870db158c Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:11:56 -0600 Subject: [PATCH 05/24] accepted change 1 Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index c2303dc30..7d001b85c 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -18,7 +18,7 @@ There are two main classes of diagnostic tests: molecular tests, which can diagn As the pandemic has evolved throughout 2020 and 2021, a variety of tests have emerged within these two categories. Molecular tests detect either viral RNA or protein in a patient sample. -Molecular tests are essential to identify infected individuals who may need treatment and whose contacts should quarantine. +Molecular tests are essential to identify infected individuals who may need to seek treatment, quarantine, and whose contacts may need to quarantine. Tests for viral RNA are done by reverse transcription (RT) of viral RNA to DNA followed by DNA amplification, usually by the polymerase chain reaction (PCR) [@DOI:10.1038/jid.2013.1]. Tests for viral proteins are most often done using detection by an antibody pair in lateral flow tests (LFTs) [@URL:https://doi.org/10.1007/s00216-010-3661-4]. Molecular tests require the viral genome sequence to develop DNA primers for viral RNA detection, or to express a viral protein for use as an antigen in antibody production. From 969d3f13ba2472f2fecbe22f55a7af84c50776da Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:12:42 -0600 Subject: [PATCH 06/24] accepted change 2 Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 7d001b85c..7e8498ba7 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -43,7 +43,7 @@ Policy discussions have also reviewed both the critical importance of diagnostic Molecular tests are used to identify distinct genomic subsequences of a viral molecule in a sample and thus to diagnose an active viral infection. An important first step is identifying which biospecimens are likely to contain the virus in infected individuals and then acquiring these samples from the patient(s) to be tested. Common sampling sources for molecular tests include nasopharyngeal cavity samples, such as throat washes, throat swabs, and saliva [@doi:10/ggp4qx], and stool samples [@doi:10.1002/jmv.25742]. -Once a sample is acquired from a patient, molecular tests detect SARS-CoV-2 based on the presence of either (1) viral nucleic acids and (2) viral protein. +Once a sample is acquired from a patient, molecular tests detect SARS-CoV-2 based on the presence of either (1) viral nucleic acids or (2) viral protein. #### Nucleic acid tests When testing for RNA from viruses like SARS-CoV-2, the first step involves pre-processing in order to create complimentary DNA (cDNA) from the RNA sample using reverse transcription (RT). From a47457f9c82976b6324b153c09535c858de6fe51 Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:12:54 -0600 Subject: [PATCH 07/24] accepted change 3 Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 7e8498ba7..83477877b 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -122,7 +122,7 @@ Overall, these studies suggest that ddPCR is a promising tool for overcoming the ##### RT-LAMP 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. -Loop Mediated Isothermal Amplification (LAMP) is an alternative to PCR that does not require a thermal cycler [@DOI:10.1093/nar/28.12.e63]. +Loop mediated isothermal amplification (LAMP) is an alternative to PCR that does not require a thermal cycler [@DOI:10.1093/nar/28.12.e63]. LAMP is combined with reverse transcription (RT-LAMP) to enable detection of RNA. RT-LAMP pilot studies for detecion of SARS-CoV-2 were recently reviewed and meta-analyzed [@PMID:34086539]. In the meta analysis of all 2,112 samples, the cumulative sensitivity of RT-LAMP was 95.5% (CI 97.5% = 90.8-97.9%) and the cumulative specificity was 99.5% (CI 97.5% = 97.7-99.9%). From c33b6e61f1e7175574147afccd37ed352172a334 Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:13:19 -0600 Subject: [PATCH 08/24] accepted change 4 Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 83477877b..c42155b5c 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -129,7 +129,7 @@ In the meta analysis of all 2,112 samples, the cumulative sensitivity of RT-LAMP The low cost, excellent sensitity/specificity, and quick readout of RT-LAMP makes this an attractive alternative to RT-PCR. Alternative strategies like RT-LAMP are needed to bring widespread testing away from the lab and into under resourced areas. -One recent study showed that RT-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 [@url:https://www.nature.com/articles/s41598-021-95799-6]. +One study showed that RT-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 [@doi:10.1038/s41598-021-95799-6]. 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. This method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. From 7ebd97ca8595d388704740431899987f1aa68deb Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:24:40 -0600 Subject: [PATCH 09/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index c42155b5c..b480b3266 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -20,7 +20,7 @@ As the pandemic has evolved throughout 2020 and 2021, a variety of tests have em Molecular tests detect either viral RNA or protein in a patient sample. Molecular tests are essential to identify infected individuals who may need to seek treatment, quarantine, and whose contacts may need to quarantine. Tests for viral RNA are done by reverse transcription (RT) of viral RNA to DNA followed by DNA amplification, usually by the polymerase chain reaction (PCR) [@DOI:10.1038/jid.2013.1]. -Tests for viral proteins are most often done using detection by an antibody pair in lateral flow tests (LFTs) [@URL:https://doi.org/10.1007/s00216-010-3661-4]. +Tests for viral proteins are most often done using detection by an antibody pair in lateral flow tests (LFTs) [@doi:10.1007/s00216-010-3661-4]. Molecular tests require the viral genome sequence to develop DNA primers for viral RNA detection, or to express a viral protein for use as an antigen in antibody production. Serological tests detect the presence of antibodies in blood plasma samples using enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA) [@DOI:10.3389/fmolb.2021.682405]. From aedc60350b702f60364c9b018012776c60a56d6d Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:24:52 -0600 Subject: [PATCH 10/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index b480b3266..266787549 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -201,7 +201,7 @@ Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other h #### Protein tests LFT-based tests are beneficial because they can detect current infection within 30 minutes and because they can be performed without specialized equipment at low cost. -LFTs rely on detection of viral protein with an antibody. +LFTs rely on the detection of viral protein with an antibody. Often this is done with an antibody sandwich format, where one antibody containing a dye is binds at one site on the antigen, and an immobilized antibody on the strip binds at another site, allowing accumulation of the dye and the characteristic formation of a positive test line on the strip [@URL:https://doi.org/10.1007/s00216-010-3661-4]. The applications of LFTs are broad: they are routinely used for home pregnancy tests, disease detection, and even drugs of abuse detection in urine [@DOI:10.1007/s00216-008-2287-2]. Current infection detection by LFTs is needed to enable the scale and speed of testing required to stop the spread of SARS-CoV-2. From b64f8b33b0e7a382a3f78a68e87deb3d580c0e73 Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:25:13 -0600 Subject: [PATCH 11/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 266787549..7774e9396 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -202,7 +202,7 @@ Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other h #### Protein tests LFT-based tests are beneficial because they can detect current infection within 30 minutes and because they can be performed without specialized equipment at low cost. LFTs rely on the detection of viral protein with an antibody. -Often this is done with an antibody sandwich format, where one antibody containing a dye is binds at one site on the antigen, and an immobilized antibody on the strip binds at another site, allowing accumulation of the dye and the characteristic formation of a positive test line on the strip [@URL:https://doi.org/10.1007/s00216-010-3661-4]. +Often this is done with an antibody sandwich format, where one antibody containing a dye binds at one site on the antigen, and an immobilized antibody on the strip binds at another site, allowing accumulation of the dye and the characteristic formation of a positive test line on the strip [@doi:10.1007/s00216-010-3661-4]. The applications of LFTs are broad: they are routinely used for home pregnancy tests, disease detection, and even drugs of abuse detection in urine [@DOI:10.1007/s00216-008-2287-2]. Current infection detection by LFTs is needed to enable the scale and speed of testing required to stop the spread of SARS-CoV-2. Lateral flow tests were made available freely to citizens in the United Kingdom at least until December 2021 [@DOI:10.1136/bmj.n1760]. From f50dfbcab93a616c915053c67949b8decd2c3fd8 Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:25:30 -0600 Subject: [PATCH 12/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 7774e9396..0e9a1867d 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -205,7 +205,7 @@ LFTs rely on the detection of viral protein with an antibody. Often this is done with an antibody sandwich format, where one antibody containing a dye binds at one site on the antigen, and an immobilized antibody on the strip binds at another site, allowing accumulation of the dye and the characteristic formation of a positive test line on the strip [@doi:10.1007/s00216-010-3661-4]. The applications of LFTs are broad: they are routinely used for home pregnancy tests, disease detection, and even drugs of abuse detection in urine [@DOI:10.1007/s00216-008-2287-2]. Current infection detection by LFTs is needed to enable the scale and speed of testing required to stop the spread of SARS-CoV-2. -Lateral flow tests were made available freely to citizens in the United Kingdom at least until December 2021 [@DOI:10.1136/bmj.n1760]. +LFTs were made available freely to citizens in the United Kingdom at least until December 2021 [@DOI:10.1136/bmj.n1760]. A recent review surveyed the performance of LFTs for detection of current SARS-CoV-2 infection [@PMID:34407759]. This review covered 24 studies that included more than 26,000 total LFTs. From e4889bdcbf78c434d19997007e552e92a173c90c Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:25:42 -0600 Subject: [PATCH 13/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 0e9a1867d..c4d4f4eab 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -203,7 +203,7 @@ Importantly, DETECTR was 100% specific for SARS-CoV-2 and did not detect other h LFT-based tests are beneficial because they can detect current infection within 30 minutes and because they can be performed without specialized equipment at low cost. LFTs rely on the detection of viral protein with an antibody. Often this is done with an antibody sandwich format, where one antibody containing a dye binds at one site on the antigen, and an immobilized antibody on the strip binds at another site, allowing accumulation of the dye and the characteristic formation of a positive test line on the strip [@doi:10.1007/s00216-010-3661-4]. -The applications of LFTs are broad: they are routinely used for home pregnancy tests, disease detection, and even drugs of abuse detection in urine [@DOI:10.1007/s00216-008-2287-2]. +The applications of LFTs are broad; they are routinely used for home pregnancy tests, disease detection, and even drugs of abuse detection in urine [@DOI:10.1007/s00216-008-2287-2]. Current infection detection by LFTs is needed to enable the scale and speed of testing required to stop the spread of SARS-CoV-2. LFTs were made available freely to citizens in the United Kingdom at least until December 2021 [@DOI:10.1136/bmj.n1760]. From e9e66b5a494c71e43f5d96d3d8fb89b10fde6e11 Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:26:06 -0600 Subject: [PATCH 14/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index c4d4f4eab..f29a74446 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -157,7 +157,7 @@ Technology based on CRISPR (clustered regularly interspaced short palindromic re ##### CRISPR-based Detection -Technology based on CRISPR (clustered regularly interspaced short palindromic repeats) [@DOI:https://doi.org/10.1038/s41467-018-04252-2] has also been instrumental in scaling up testing protocols. +Technology based on CRISPR (clustered regularly interspaced short palindromic repeats) [@doi:10.1038/s41467-018-04252-2] has also been instrumental in scaling up testing protocols. Two CRISPR-associated nucleases, Cas12 and Cas13, have been used for nucleic acid detection. Multiple assays exploiting these nucleases have emerged as potential diagnostic tools for the rapid detection of SARS-CoV-2 genetic material and therefore SARS-CoV-2 infection. The SHERLOCK method (Specific High-sensitivity Enzymatic Reporter unLOCKing) from Sherlock Biosciences relies on Cas13a to discriminate between inputs that differ by a single nucleotide at very low concentrations [@doi:10.1126/science.aam9321]. From fad67398049d8da480ce8a07ec8c703acf33f2b1 Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:26:28 -0600 Subject: [PATCH 15/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index f29a74446..8c5ca499d 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -137,7 +137,7 @@ The authors break down the sensitivity of their test according to the cycle thre The performance is worse when considering all RT-PCR positive samples (including those with Ct values between 32-40). 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 [@URL:https://doi.org/10.1101/2020.02.26.20028373]. To determine assay sensitivity, serial tenfold dilutions of _in vitro_ transcribed N-gene RNA standard (IVT RNA) were tested using quantities from 10^5 copies down to 10 copies. -The assay readout is the color of dye changing from red to yellow due to binding to the DNA product over 30 minutes. +The assay readout is the color of the dye changing from pink to yellow due to binding to the DNA product over 30 minutes. The RT-LAMP assay was then applied to clinical nasopharyngeal samples. For viral loads above 100 copies of genomic RNA, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1% from purified RNA. The direct assay of saliva by RT-LAMP had a sensitivity of 85% (CI 70-93%). From b9132a10bdf767001536b5e882d1882ec7bbc41d Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Sun, 2 Jan 2022 14:56:12 -0600 Subject: [PATCH 16/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 1 + 1 file changed, 1 insertion(+) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 8c5ca499d..5f2bebd9d 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -135,6 +135,7 @@ It could be applied for screening, diagnostics, or as a definitive test for peop This method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. The authors break down the sensitivity of their test according to the cycle threshold (Ct) value from RT-PCR of the same samples; RT-LAMP performs at 100% sensitivity for samples with a Ct from RT-PCR of 32 or less. The performance is worse when considering all RT-PCR positive samples (including those with Ct values between 32-40). +However, there is some evidence suggesting that samples obtained from individuals that achieve Ct values >30 measured using RT-PCR tend to be less infective that those that record a Ct value <30 [@doi:10.1016/j.jiph.2021.08.013; @doi:10.1017/ice.2021.132; @doi:10.1093/cid/ciaa619], and so RT-LAMP is still a useful diagnostic tool. 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 [@URL:https://doi.org/10.1101/2020.02.26.20028373]. To determine assay sensitivity, serial tenfold dilutions of _in vitro_ transcribed N-gene RNA standard (IVT RNA) were tested using quantities from 10^5 copies down to 10 copies. The assay readout is the color of the dye changing from pink to yellow due to binding to the DNA product over 30 minutes. From e3d17a88ebae4ca6873f5be18505aabe257bb617 Mon Sep 17 00:00:00 2001 From: jessegmeyerlab <65188012+jessegmeyerlab@users.noreply.github.com> Date: Wed, 5 Jan 2022 07:04:25 -0600 Subject: [PATCH 17/24] Update content/10.diagnostics.md Co-authored-by: Ronan Lordan <62627112+RLordan@users.noreply.github.com> --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 5f2bebd9d..d01788504 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -136,7 +136,7 @@ This method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA sa The authors break down the sensitivity of their test according to the cycle threshold (Ct) value from RT-PCR of the same samples; RT-LAMP performs at 100% sensitivity for samples with a Ct from RT-PCR of 32 or less. The performance is worse when considering all RT-PCR positive samples (including those with Ct values between 32-40). However, there is some evidence suggesting that samples obtained from individuals that achieve Ct values >30 measured using RT-PCR tend to be less infective that those that record a Ct value <30 [@doi:10.1016/j.jiph.2021.08.013; @doi:10.1017/ice.2021.132; @doi:10.1093/cid/ciaa619], and so RT-LAMP is still a useful diagnostic tool. -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 [@URL:https://doi.org/10.1101/2020.02.26.20028373]. +Various combinations of reagents are available, but one example of reagents being used are the WarmStart Colorimetric LAMP 2X Master Mix with a set of six primers developed previously by Zhang et al. [@doi:10.1101/2020.02.26.20028373]. To determine assay sensitivity, serial tenfold dilutions of _in vitro_ transcribed N-gene RNA standard (IVT RNA) were tested using quantities from 10^5 copies down to 10 copies. The assay readout is the color of the dye changing from pink to yellow due to binding to the DNA product over 30 minutes. The RT-LAMP assay was then applied to clinical nasopharyngeal samples. From 4f8d4298c5a8e39b35bc5b3f6af134038e22e7f7 Mon Sep 17 00:00:00 2001 From: Ronan Lordan <62627112+RLordan@users.noreply.github.com> Date: Thu, 6 Jan 2022 13:08:53 -0500 Subject: [PATCH 18/24] Update content/10.diagnostics.md --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 96e873f8d..06d9a79e7 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -134,7 +134,7 @@ This test aims to bring the sensitivity of nucleic acid detection to the point o It could be applied for screening, diagnostics, or as a definitive test for people who are positive based on lateral flow tests. This method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. The authors break down the sensitivity of their test according to the cycle threshold (Ct) value from RT-PCR of the same samples; RT-LAMP performs at 100% sensitivity for samples with a Ct from RT-PCR of 32 or less. -The performance is worse when considering all RT-PCR positive samples (including those with Ct values between 32-40). +The performance is worse when considering all RT-PCR positive samples (including those with C~t~ values between 32-40). However, there is some evidence suggesting that samples obtained from individuals that achieve Ct values >30 measured using RT-PCR tend to be less infective that those that record a Ct value <30 [@doi:10.1016/j.jiph.2021.08.013; @doi:10.1017/ice.2021.132; @doi:10.1093/cid/ciaa619], and so RT-LAMP is still a useful diagnostic tool. Various combinations of reagents are available, but one example of reagents being used are the WarmStart Colorimetric LAMP 2X Master Mix with a set of six primers developed previously by Zhang et al. [@doi:10.1101/2020.02.26.20028373]. To determine assay sensitivity, serial tenfold dilutions of _in vitro_ transcribed N-gene RNA standard (IVT RNA) were tested using quantities from 10^5 copies down to 10 copies. From a8300a68f574223f2bc24afde5112a049a35506b Mon Sep 17 00:00:00 2001 From: Ronan Lordan <62627112+RLordan@users.noreply.github.com> Date: Thu, 6 Jan 2022 13:09:12 -0500 Subject: [PATCH 19/24] Update content/10.diagnostics.md Co-authored-by: HM Rando --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 06d9a79e7..4f193138b 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -24,7 +24,7 @@ Tests for viral proteins are most often done using detection by an antibody pair Molecular tests require the viral genome sequence to develop DNA primers for viral RNA detection, or to express a viral protein for use as an antigen in antibody production. Serological tests detect the presence of antibodies in blood plasma samples using enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA) [@DOI:10.3389/fmolb.2021.682405]. -Because vaccines are based on the viral spike protein, to distinguish past infection from vaccination, serology tests look for antibodies that bind the nucleocapsid protein of the SARS-CoV-2 virus[@doi:10.1093/infdis/jiaa273]. +Because vaccines are based on the viral spike protein, to distinguish past infection from vaccination, serology tests look for antibodies that bind the nucleocapsid protein of the SARS-CoV-2 virus [@doi:10.1093/infdis/jiaa273]. Serological tests are useful for collecting population-level information for epidemiological analysis, as they can be used to estimate the extent of the infection in a given area. Thus, serological tests may be useful to better understand the percent of cases that manifest as severe versus mild and for guiding public health and economic decisions regarding resource allocation and counter-disease measures. From 8d242336ed19148bae582d74af8d842c24f1ae42 Mon Sep 17 00:00:00 2001 From: Ronan Lordan <62627112+RLordan@users.noreply.github.com> Date: Thu, 6 Jan 2022 13:09:19 -0500 Subject: [PATCH 20/24] Update content/10.diagnostics.md Co-authored-by: HM Rando --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 4f193138b..9d7468522 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -45,7 +45,7 @@ An important first step is identifying which biospecimens are likely to contain Common sampling sources for molecular tests include nasopharyngeal cavity samples, such as throat washes, throat swabs, and saliva [@doi:10/ggp4qx], and stool samples [@doi:10.1002/jmv.25742]. Once a sample is acquired from a patient, molecular tests detect SARS-CoV-2 based on the presence of either (1) viral nucleic acids or (2) viral protein. -#### Nucleic acid tests +#### Nucleic Acid Tests When testing for RNA from viruses like SARS-CoV-2, the first step involves pre-processing in order to create complimentary DNA (cDNA) from the RNA sample using reverse transcription (RT). The second step involves the amplification of a region of interest in the cDNA using successive cycles of heating and cooling. Depending on the application, amplification is achieved using variations of the polymerase chain reaction (PCR). From 2c0363a1303d5bc8099f85be3b33f86a6da20097 Mon Sep 17 00:00:00 2001 From: Ronan Lordan <62627112+RLordan@users.noreply.github.com> Date: Thu, 6 Jan 2022 13:09:26 -0500 Subject: [PATCH 21/24] Update content/10.diagnostics.md Co-authored-by: HM Rando --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 9d7468522..d7df25c1c 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -51,7 +51,7 @@ The second step involves the amplification of a region of interest in the cDNA u Depending on the application, amplification is achieved using variations of the polymerase chain reaction (PCR). Nucleic acid tests differ primarily in detection methods, which are detailed below. -##### SEQUENCING +##### Sequencing Some tests use the results of the PCR itself to determine whether the pathogen is present, but in other cases, it may be necessary to sequence the amplified DNA. Sequencing requires an additional sample pre-processing step called library preparation. Library preparation is the process of preparing the sample for sequencing, typically by fragmenting the sequences and adding adapters [@doi:10.1016/j.biotechadv.2020.107537]. From 702c57d8d956ab33336b591b22bf9f8b0c41a3e0 Mon Sep 17 00:00:00 2001 From: Ronan Lordan <62627112+RLordan@users.noreply.github.com> Date: Thu, 6 Jan 2022 13:09:34 -0500 Subject: [PATCH 22/24] Update content/10.diagnostics.md Co-authored-by: HM Rando --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index d7df25c1c..205a5460b 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -133,7 +133,7 @@ One study showed that RT-LAMP is effective for detection of SARS-CoV-2 with exce 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. This method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. -The authors break down the sensitivity of their test according to the cycle threshold (Ct) value from RT-PCR of the same samples; RT-LAMP performs at 100% sensitivity for samples with a Ct from RT-PCR of 32 or less. +The authors break down the sensitivity of their test according to the cycle threshold (C~t~) value from RT-PCR of the same samples; RT-LAMP performs at 100% sensitivity for samples with a C~t~ from RT-PCR of 32 or less. The performance is worse when considering all RT-PCR positive samples (including those with C~t~ values between 32-40). However, there is some evidence suggesting that samples obtained from individuals that achieve Ct values >30 measured using RT-PCR tend to be less infective that those that record a Ct value <30 [@doi:10.1016/j.jiph.2021.08.013; @doi:10.1017/ice.2021.132; @doi:10.1093/cid/ciaa619], and so RT-LAMP is still a useful diagnostic tool. Various combinations of reagents are available, but one example of reagents being used are the WarmStart Colorimetric LAMP 2X Master Mix with a set of six primers developed previously by Zhang et al. [@doi:10.1101/2020.02.26.20028373]. From 12e980822a1356796c73bcfb63adbccb171129ac Mon Sep 17 00:00:00 2001 From: Ronan Lordan <62627112+RLordan@users.noreply.github.com> Date: Thu, 6 Jan 2022 13:09:40 -0500 Subject: [PATCH 23/24] Update content/10.diagnostics.md Co-authored-by: HM Rando --- content/10.diagnostics.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/content/10.diagnostics.md b/content/10.diagnostics.md index 205a5460b..09c34f0bd 100644 --- a/content/10.diagnostics.md +++ b/content/10.diagnostics.md @@ -135,7 +135,7 @@ It could be applied for screening, diagnostics, or as a definitive test for peop This method was benchmarked against RT-PCR using 177 human nasopharyngeal RNA samples, of which 126 were COVID positive. The authors break down the sensitivity of their test according to the cycle threshold (C~t~) value from RT-PCR of the same samples; RT-LAMP performs at 100% sensitivity for samples with a C~t~ from RT-PCR of 32 or less. The performance is worse when considering all RT-PCR positive samples (including those with C~t~ values between 32-40). -However, there is some evidence suggesting that samples obtained from individuals that achieve Ct values >30 measured using RT-PCR tend to be less infective that those that record a Ct value <30 [@doi:10.1016/j.jiph.2021.08.013; @doi:10.1017/ice.2021.132; @doi:10.1093/cid/ciaa619], and so RT-LAMP is still a useful diagnostic tool. +However, there is some evidence suggesting that samples obtained from individuals that achieve C~t~ values >30 measured using RT-PCR tend to be less infective that those that record a C~t~ value <30 [@doi:10.1016/j.jiph.2021.08.013; @doi:10.1017/ice.2021.132; @doi:10.1093/cid/ciaa619], so RT-LAMP is still a useful diagnostic tool. Various combinations of reagents are available, but one example of reagents being used are the WarmStart Colorimetric LAMP 2X Master Mix with a set of six primers developed previously by Zhang et al. [@doi:10.1101/2020.02.26.20028373]. To determine assay sensitivity, serial tenfold dilutions of _in vitro_ transcribed N-gene RNA standard (IVT RNA) were tested using quantities from 10^5 copies down to 10 copies. The assay readout is the color of the dye changing from pink to yellow due to binding to the DNA product over 30 minutes. From e0e44d5d03451bbb17bec839bbdb0f84f4c146c9 Mon Sep 17 00:00:00 2001 From: HM Rando Date: Thu, 6 Jan 2022 14:00:58 -0500 Subject: [PATCH 24/24] Update metadata.yaml --- content/metadata.yaml | 17 +++++++++++++++++ 1 file changed, 17 insertions(+) diff --git a/content/metadata.yaml b/content/metadata.yaml index 3006b8b29..d9f90b268 100644 --- a/content/metadata.yaml +++ b/content/metadata.yaml @@ -1387,3 +1387,20 @@ authors: order: 1 contributions: - Writing - Review & Editing + - + github: jessegmeyerlab + name: Jesse G. Meyer + initials: JGM + orcid: 0000-0003-2753-3926 + email: jessegmeyer@gmail.com + affiliations: + - Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America + coi: + string: "None" + lastapproved: !!str 2022-01-06 + manuscripts: + diagnostics: + order: 1 + contributions: + - Writing - Review & Editing + - Writing - Original Draft