Update 10.diagnostics.md

added minimum of LFT section under molecular tests, minor edits elsewhere

content/10.diagnostics.md

@@ -13,22 +13,21 @@ For instance, test-trace-isolate procedures were an early cornerstone of many na
 <!-- To Do: possibly add text on what goes into developing new diagnostics?-->
 
 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]. 
 <!-- To Do: How did we discover the identity of these to start binding them?-->
-As the pandemic has evolved throughout 2020 and 2021, a variety of technological implementations have emerged within these two categories.
-
 <!-- To Do: Discuss HCoV broadly and the potential risk of detecting other coronaviruses-->
 <!-- To Do: Trade offs in cost vs precision (throughput)-->
 <!-- To Do: Discuss viral load vs contagiousness vs symptoms vs antibody development? -->
@@ -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]). 
 <!-- To Do: What is the advantage of the sequencing-based tests?-->
 
 ##### 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.
 
-<!--To do: section on #### Protein tests
+#### 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 are routinely used to detect a variety of diseases and even drugs of abuse in urine (citations).-->
-
-
+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).
+<!-- To do: examples of specific studies evaluating LFTs for covid detection -->
 
 #### Limitations of Molecular Tests