PHLN guidance for serological testing in COVID-19
Public Health Laboratory Network Guidance for serological testing in COVID-19Revision historyVersionDate Endorsed by PHLNRevision note1.33 September 2020Update to clarify language and add table of comments for IgG only assays. 1.220 July 2020Update to strengthen the direction to conduct confirmatory testing for positive serology results.1.122 June 2020Initial document High-quality serological assays are now available in Australia for detection of SARS-CoV-2 (the virus that causes COVID-19) specific antibodies. Before widespread deployment of these assays, appropriate frameworks are required to inform:who should be testedwhich assays and confirmatory protocols to use, and how to report test results.BackgroundOne of the most important ways to prevent and control COVID-19 is timely, scalable and accurate diagnostic testing. Diagnostic testing plays a critical role in defining the epidemiology of the disease, informing case and contact management, and ultimately in reducing viral transmission. To date, laboratory testing has largely comprised detection of SARS-CoV-2 using reverse-transcriptase polymerase chain reaction (RT-PCR) assays. More recently, a range of serological tests have become available. Situations where serological testing may be of possible use include:Where the result will influence individual or outbreak management, testing patients who have had symptoms consistent with COVID-19 but are RT-PCR negative OR;were not tested OR; have unexpected positive or inconclusive results on RT-PCR assaysSeroepidemiological studies to define the degree of population infectionSurveillance of frontline healthcare workers to define potential occupational infectionConvalescent patients, for plasma donation Patients who may have been, or are, part of an outbreak investigationEstimating timing of infection to help define the likely infectious period where this is not evident from clinical symptoms or exposure history.Serological tests rely on detection of specific anti-SARS-CoV-2 antibodies (IgM, IgA, IgG or total antibody) in patient serum, plasma or whole blood. Determining the optimal antigenic epitopes to maximise sensitivity but minimise cross-reactivity, particularly against other human coronaviruses, has meant the development of high-quality serological testing has been slower than molecular-based diagnostics. In addition, as antibody responses take time to develop, serological assays evaluated to date have typically demonstrated adequate sensitivity 14 days after symptom onset, limiting their utility in the clinical and public health management of acute infections. Occasionally the serological response may take longer to develop (28 days) and approximately 5-10% of individuals may not develop antibodies detectable on current test platforms. ADDIN EN.CITE <EndNote><Cite><Author>Tang</Author><Year>2020</Year><RecNum>8622</RecNum><DisplayText><style face="superscript">1</style></DisplayText><record><rec-number>8622</rec-number><foreign-keys><key app="EN" db-id="d0zft2ttw92av6eetwq5aved29x5xfd520w9" timestamp="1592374862">8622</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Tang, M. S.</author><author>Hock, K. G.</author><author>Logsdon, N. M.</author><author>Hayes, J. E.</author><author>Gronowski, A. M.</author><author>Anderson, N. W.</author><author>Farnsworth, C. W.</author></authors></contributors><auth-address>Department of Pathology & Immunology. Washington University in St. Louis School of Medicine. St. Louis, MO.
Department of Laboratories, Barnes Jewish Hospital Department of Laboratories. St. Louis, MO.</auth-address><titles><title>Clinical Performance of Two SARS-CoV-2 Serologic Assays</title><secondary-title>Clin Chem</secondary-title></titles><periodical><full-title>Clin Chem</full-title></periodical><edition>2020/05/14</edition><keywords><keyword>Covid-19</keyword><keyword>SARS-CoV-2</keyword><keyword>Serology</keyword></keywords><dates><year>2020</year><pub-dates><date>May 13</date></pub-dates></dates><isbn>0009-9147 (Print)
0009-9147</isbn><accession-num>32402061</accession-num><urls></urls><custom2>PMC7239232</custom2><electronic-resource-num>10.1093/clinchem/hvaa120</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>1Broadly, serological tests for COVID-19 are tests that:Approved specimen collection personnel perform at the point-of care, Pathologists and medical laboratory scientists perform in routine diagnostic laboratories (e.g. commercial enzyme immunoassays), orPathologists and medical laboratory scientists perform in specialised reference laboratories (e.g. viral neutralisation assays, microsphere immunoassays and immunofluorescent assays). Despite the increase in availability of serological assays, many have undergone limited validation, making selecting assays difficult. In addition, there remain fundamental knowledge gaps in the interpretation of serological test results, including:Whether antibody detection correlates with protective immunity to reinfectionWhether antibody detection correlates with lack of infectiousness to othersThe amount and duration of antibody response, particularly in mild or asymptomatic infectionThe interpretation of serological assays in low prevalence settings (i.e. identification of true positive results).Who should be tested?In general, clinicians should only request pathology testing if it is likely to contribute to the management of the individual and/or public health. This minimises the harms associated with unanticipated test results and ensures responsible use of healthcare resources.Given the time lag from symptom onset to detectable antibody, serological assays have no role in the detection of acute COVID-19 infection. Specimens may, however, be collected and stored for later testing if required. In the first instance, RT-PCR tests should be requested for acute diagnosis of COVID-19 as it is the gold-standard. ADDIN EN.CITE <EndNote><Cite><Author> (2020). Last accessed 14th April, 2020.</Author><RecNum>8598</RecNum><DisplayText><style face="superscript">2</style></DisplayText><record><rec-number>8598</rec-number><foreign-keys><key app="EN" db-id="d0zft2ttw92av6eetwq5aved29x5xfd520w9" timestamp="1587616922">8598</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Public Health Laboratory Network Australia. Statement on point-of-care serology testing for SARS-CoV-2. Available at: (2020). Last accessed 14th April, 2020.</author></authors></contributors><titles></titles><dates></dates><urls></urls></record></Cite></EndNote>2 However, serology may be used to support the diagnosis in cases where RT-PCR results are inconclusive. For further guidance on when to consider serology to support the diagnosis of COVID-19, see the PHLN guidance on laboratory testing for SARS-CoV-2.If a clinician requests serology they should provide the date of onset of symptoms (where possible) to enable accurate interpretation of the serological result. Approved specimen collection personnel should perform serological testing for COVID-19 with guidance from the local laboratory. Serological testing before two weeks from the onset of symptoms may result in false negative results. Occasionally the serological response may take longer to develop and approximately 5-10% of individuals may not develop antibodies. ADDIN EN.CITE <EndNote><Cite><Author>Tang</Author><Year>2020</Year><RecNum>8622</RecNum><DisplayText><style face="superscript">1</style></DisplayText><record><rec-number>8622</rec-number><foreign-keys><key app="EN" db-id="d0zft2ttw92av6eetwq5aved29x5xfd520w9" timestamp="1592374862">8622</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Tang, M. S.</author><author>Hock, K. G.</author><author>Logsdon, N. M.</author><author>Hayes, J. E.</author><author>Gronowski, A. M.</author><author>Anderson, N. W.</author><author>Farnsworth, C. W.</author></authors></contributors><auth-address>Department of Pathology & Immunology. Washington University in St. Louis School of Medicine. St. Louis, MO.
Department of Laboratories, Barnes Jewish Hospital Department of Laboratories. St. Louis, MO.</auth-address><titles><title>Clinical Performance of Two SARS-CoV-2 Serologic Assays</title><secondary-title>Clin Chem</secondary-title></titles><periodical><full-title>Clin Chem</full-title></periodical><edition>2020/05/14</edition><keywords><keyword>Covid-19</keyword><keyword>SARS-CoV-2</keyword><keyword>Serology</keyword></keywords><dates><year>2020</year><pub-dates><date>May 13</date></pub-dates></dates><isbn>0009-9147 (Print)
0009-9147</isbn><accession-num>32402061</accession-num><urls></urls><custom2>PMC7239232</custom2><electronic-resource-num>10.1093/clinchem/hvaa120</electronic-resource-num><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>1 If the clinical suspicion is high, PHLN recommends testing out to 28 days post-presumed exposure or symptom onset (if available). As more data become available regarding immune responses to SARS-CoV-2, the clinical indications for serological testing may become more evident and broader.PHLN recommends that the decision to perform SARS-CoV-2 serological testing is made with all relevant information, including date of symptom onset, and in consultation with:public health, and/or the treating clinician, and the clinical microbiologist.How to test?Robust validation of serological assays for COVID-19 is essential to assess their accuracy in the Australian setting. As is the identification of optimal approaches to confirming true positive and negative tests. The prevalence of COVID-19 varies in different jurisdictions within Australia. The positive predictive value of serology testing is inversely proportionate to disease prevalence in the tested population (Figure 1). Testing of those without epidemiological risk factors and/or clinical features of COVID-19 may lead to a high proportion of false positive results. This could occur even with a highly specific laboratory test. In such situations, PHLN recommends supplemental or confirmatory testing.Figure 1. False positive rates of serological assays stratified by population prevalence of disease and assay specificity and sensitivity. Coloured lines denote specificity.Current CDNA guidelines state that seroconversion, or a significant (e.g. four-fold or greater) rise in either neutralising or IgG antibody level is definitive laboratory evidence of SARS-CoV-2 infection. Whereas, detection of IgG antibody in a single specimen from a person with suspected COVID- 19 is only suggestive evidence of SARS-CoV-2 infection. Pathologists and medical laboratory scientists should consider this in conjunction with clinical and epidemiological evidence when interpreting results.3 This distinction is to convey the inherent uncertainty associated with single reactive results in the context of low population prevalence, and limits to the specificity of currently available serological tests.At present, based on published sensitivity and specificity data of commercially available serological assays, there is no single serological assay that:has sufficient sensitivity to detect all cases – i.e. minimise false negatives, andhas sufficient specificity such that all positives are true – i.e. minimise non-specific reactivity or false positives.Accordingly, in the absence of seroconversion or a significant rise in antibody level between two specimens, low-prevalence settings will require a combination of testing approaches. Specific approaches may vary according to jurisdictional access to high specificity assays, particularly virus neutralisation assays. At present, neutralisation assays are only available in three jurisdictions (Victoria, NSW and QLD). Currently, no Western Blot assays are available for confirmatory testing.Detection of a positive IgM or IgA antibody without IgG detection is not sufficient evidence of recent COVID-19 (see suggested comments below). PHLN recommends the clinician requests a repeat specimen collection to look for IgG seroconversion.In the event of a single IgG positive specimen (with or without IgM and/or IgA antibody) from a suspected case of COVID-19 with supportive clinical and epidemiological evidence, the clinician should request a follow-up sample to look for a significant rise in antibody level.PHLN recommends applying one of the two following approaches to help confirm the presence of anti-SARS-CoV-2 antibodies, depending on jurisdictional capabilities, if: the follow-up specimen fails to demonstrate a significant rise in antibody level, or in the event of a single IgG equivocal result, or reactive result in a person when using a commercially available enzyme immunoassay (or another validated test). This is without supportive clinical and epidemiological evidence (or in the absence of access to such information)Approach 1: Request a follow-up sample (see below). Retest the initial sample using an alternative commercially-available enzyme immunoassay (or other validated test) with comparable or greater sensitivity/specificity, ideally targeting a different antigen to the initial test.Approach 2: Request a follow-up sample (see below). Retest the initial sample using a virus neutralisation assay or other alternate format reference laboratory antibody test, such as immunofluorescent assay or microsphere immunoassay.How to report?Pathologists and medical laboratory scientists should interpret serological test results in association with the patient’s clinical and epidemiological information and other laboratory results. When serological results for COVID-19 are reported, it is important to understand:the analytical sensitivity and specificity of the assay; and the likely prevalence of the patient population being tested.PHLN suggests using the following comments as a framework for reporting serological tests for COVID-19. PHLN recognises that individual laboratories will tailor comments according to their local situation.SARS-CoV-2 IgGSARS-CoV-2 IgM/ IgAInterpretive commentNon-reactiveNon-reactiveNo serological evidence of infection with SARS-CoV-2. Please submit a further sample in 10-14 days if recent infection is suspected, to assist in confirming or excluding infection.Non-reactiveEquivocalPossible recent infection with SARS-CoV-2 or nonspecific reactivity. Please submit a further sample in 10-14 days if recent infection is suspected, to assist in confirming or excluding infection.Non-reactiveREACTIVEPossible recent infection with SARS-CoV-2 or nonspecific reactivity. Please submit a further sample in 10-14 days if recent infection is suspected, to assist in confirming or excluding infection. EquivocalNon-reactivePossible recent infection with SARS-CoV-2, distant past infection or nonspecific reactivity. Please submit a further sample in 10-14 days to assist in confirming or excluding infectionEquivocalEquivocalPossible recent infection with SARS-CoV-2 or nonspecific reactivity. Please submit a further sample in 10-14 days if recent infection is suspected, to assist in confirming or excluding infection.EquivocalREACTIVEPossible recent infection with SARS-CoV-2. Please submit a further sample in 10-14 days to assist in confirming or excluding infection if recent infection is suspected, to assist in confirming or excluding infection.REACTIVENon-reactiveConsistent with recent or past infection with SARS-CoV-2. This result should be considered with clinical and other information. Please submit a further sample in 10-14 days if recent infection is suspected.REACTIVEEquivocalConsistent with recent infection with SARS-CoV-2. This result should be considered with clinical and other information. Please submit a further sample in 10-14 days to monitor antibody response.REACTIVEREACTIVEConsistent with recent infection with SARS-CoV-2. This result should be considered with clinical and other information. Please submit a further sample in 10-14 days to monitor antibody response.In laboratories where only IgG testing is used (i.e. not with IgM and / or IgA), PHLN suggest using the following comments:SARS-CoV-2 IgGInterpretive commentNon-reactiveNo serological evidence of infection with SARS-CoV-2. Please submit a further sample in 10-14 days if recent infection is suspected, to assist in confirming or excluding infection.EquivocalPossible recent or past infection with SARS-CoV-2, or nonspecific reactivity. Please submit a further sample in 10-14 days to assist in confirming or excluding infectionREACTIVEConsistent with recent or past infection with SARS-CoV-2. This result should be considered with clinical and other information. Please submit a further sample in 10-14 days to monitor antibody level.PHLN suggests the following comments can be used, if a laboratory uses two assays. Noting, if referred to a reference laboratory, the reference result should inform the overall interpretation instead.SARS-CoV-2 IgG(antigen 1)SARS-CoV-2 IgG(antigen 2)Interpretive commentNon-reactiveNo serological evidence of infection with SARS-CoV-2. Please submit a further sample in 10-14 days if recent infection is suspected, to assist in confirming or excluding infection.Reactive/EquivocalNon-reactiveResults discordant. The initial positive/equivocal result could not be confirmed using an alternative assay with comparable sensitivity/specificity, targeting a different antigen. In a low prevalence setting, a false positive screening result is likely. Alternatively recent or past infection with SARS-CoV-2 is possible and interpretation requires consideration of clinical and epidemiological evidence of likely recent infection with SARS-CoV-2. Referral to a reference laboratory may be indicatedPlease submit a further sample in 10-14 days to assist in confirming or excluding infection. Reactive/EquivocalEquivocal/ReactiveConsistent with probable recent or past infection with SARS-CoV-2. The initial positive/equivocal result was confirmed using an alternative assay with comparable sensitivity and specificity, targeting a different antigen. This increases the positive predictive value of this result, however final interpretation should consider the supportive clinical and epidemiological evidence of likely infection with SARS-CoV-2. Referral to a reference laboratory may be indicated.Please submit a further sample in 10-14 days to assist in confirming or excluding infection.ReactiveReactiveConsistent with recent or past infection with SARS-CoV-2. The initial positive result was confirmed using an alternative assay with comparable sensitivity and specificity, targeting a different antigen. This increases the positive predictive value of this result, however final interpretation should consider the supportive clinical and epidemiological evidence of likely infection with SARS-CoV-2.Please submit a further sample in 10-14 days to assist in confirming or excluding infection.In addition, PHLN suggests laboratories use the following comments (depending on local context) when reporting all serological test results for COVID-19:The detection of IgG antibody to SARS-CoV-2 has been confirmed by a second antibody assayThe detection of IgG antibody to SARS-CoV-2 has not been confirmed by a second antibody assayIf acute infection is suspected, please submit a respiratory sample for PCR, advise the patient to isolate and liaise with local public health authoritiesSerological testing is not appropriate for the acute diagnosis of COVID-19Testing is for antibodies to SARS-CoV-2, the causative agent of COVID-19Results should be interpreted in association with all other information (clinical, epidemiological and laboratory) on the patientReports based on this assay are not currently NATA accredited.Laboratories offering SARS-CoV-2 serology should be NATA accredited following submission of validation or verification data and participate in a recognised Quality Assurance Programme such as the one offered by the Royal College of Pathologists Australasia Quality Assurance Program (RCPAQAP).References ADDIN EN.REFLIST 1.Tang MS, Hock KG, Logsdon NM, et al. Clinical Performance of Two SARS-CoV-2 Serologic Assays. Clin Chem 2020.2. (2020). Last accessed 14th April, 2020. PHLNASop-o-cstfS-C-Aa.3.CDNA. $File/interim-COVID-19-SoNG-v2.5.pdf. 2020. ................
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