WHO knuser Norges bruk av PCR-testen – den skal kun brukes som hjelpemiddel31 min read

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WHO knuser Norges bruk av PCR-testen – den skal kun brukes som hjelpemiddel31 min read

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WHO knuser Norges bruk av PCR-testen – den skal kun brukes som hjelpemiddel (se topp-foto, her, CEPI lansert av Gro Harlem og den norske stat).

Dermed: Er bruken av PCR-testen i Norge i dag villedende og ulovlig? Har befolkning og bedrifter som har blitt nedstengt på grunn av PCR-testen krav på erstatning?

Vi gjør oppmerksom på ny informasjon fra WHO (Verdens helsorganisasjon), datert 13. januar 2021: «WHO Information Notice for IVD Users 2020/05 Nucleic acid testing (NAT) technologies that use polymerase chain reaction (PCR) for detection of SARS-CoV-2»

Informasjonen erstatter den som er gitt tidligere.

Blant annet heter det nå:

«De fleste PCR-analyser er indikert som et hjelpemiddel for diagnose, og derfor må helsepersonell vurdere ethvert resultat i kombinasjon med tidspunktet for prøvetaking, prøvetype, analysespesifikasjoner, kliniske observasjoner, pasienthistorie, bekreftet status for eventuelle kontakter og epidemiologisk informasjon.»

Videre:

«WHO-veiledning Diagnostisk testing for SARS-CoV-2 sier at det er nødvendig med nøye tolkning av svake positive resultater (1). Syklusterskelen (Ct) som er nødvendig for å oppdage virus er omvendt proporsjonal med pasientens virusbelastning. Hvis testresultatene ikke samsvarer med den kliniske presentasjonen, bør en ny prøve tas og testes på nytt med samme eller annen NAT-teknologi.»

«WHO minner IVD-brukere om at sykdomsutbredelse endrer den prediktive verdien av testresultatene; ettersom sykdomsutbredelsen avtar, øker risikoen for falske positive (2). Dette betyr at sannsynligheten for at en person som har et positivt resultat (SARS-CoV-2 oppdaget) virkelig er infisert med SARS-CoV-2, avtar etter hvert som prevalensen avtar, uavhengig av den påståtte spesifisiteten.»

«Hvis testresultatene ikke samsvarer med den kliniske presentasjonen, bør en ny prøve tas og testes på nytt med samme eller annen NAT-teknologi.»

Informasjonen holdes i et byråkratisk-medisinsk fagspråk. Men det kan ikke være tvil at det som faktisk blir uttrykt er følgende:

PCR-testen brukes helt feil i Norge.

Vi ber om svar på våre spørsmål så fort som praktisk mulig (etter forvaltningsloven osv).

A. Hvis ikke testresultatene samsvarer med klinisk presentasjoner, så må en gjøre grundigere undersøkelser.

Spørsmål:

1. Er dette gjort så langt i Norge?

2. Vil det blir gjort framover?

3. Er det vitenskapelig bevist at SARS-CoV-2 finnes ved at det er isolert? (se informasjon fra CDC lenger ned)

4. Hvordan er det med testresultatene til det engelske muterte viruset – er det gjort i overensstemmelse med denne WHO-oppdateringen?

5. Hvis den ikke er gjort det, hva slags grunnlag har man da for siste nedstenging/tiltak i 25 kommuner (inkludert Oslo)?

B. Det heter at PCR-testen «er et hjelpemiddel for diagnose» og ethvert resultat må vurderes i kombinasjon med kliniske observasjoner, pasienthistorie osv. Men til nå i Norge har PCR-testen etter det vi forstår alene blitt benyttet som bevis for smitte og indikasjon på sykdom.

Spørsmål:

1. Vil denne klare feilen blir rettet opp – og i så fall hvordan?

2. Vil bedrifter og andre som har lidd økonomisk tap på grunn av feilbruk av PCR-test få erstatning?

3. Kan PCR-testen i det hele tatt brukes som bevis for smitte eller sykdom?

4. Vil Folkehelseinstituttet, med Helsedirektoratet og Helse- og omsorgsdepartementet, endre på testkriteriene?

5. Vil Folkehelseinstituttet, med Helsedirektoratet og Helse- og omsorgsdepartementet, endre på sine råd til regjeringen når det gjelder nedstengning og andre smitteverntiltak?

6. Vil Folkehelseinstituttet, med Helsedirektoratet og Helse- og omsorgsdepartementet, endre på sine råd til kommuner, kommuneoverleger, helseforetak, helsepersonell, barnehager, skoler og næringsliv?

7. Vil den mentale belastningen små og store har opplevd fra 12. mars i fjor kunne oppveies?

8. Er innføringen av vaksinen basert på feil grunnlag – og må vaksinen/vaksinene trekkes omgående tilbake?

9. Er det sammenhengen mellom vaksineringsønske og det store fokus på smittetall fra en test som brukes feil?

C. Sykluser oppgis av WHO å være nødvendig for å oppdage virus.

Spørsmål:

1. Hvor mange sykluser benyttes ved tester i Norge?

2. Hvor mange av disse gir falske positive?

3. Kan antall sykluser påvirke testen i så stor grad at regjeringen går til langt strengere tiltak enn nødvendig?

4. Er det slik at Folkehelseinstituttet gjør uavhengige undersøkelser av virus og test eller er informasjonen dere har basert på organ som WHO, Rockefeller Foundation, EU med mer?

5. Kan PCR-feil gi gale premisser for planer om koronapass og digitale adgangstegn?

D. På generelt grunnlag om PCR-testen:

Spørsmål:

1. Kan testen skille mellom virus og fragmenter av virus?

2. Hvis svaret er nei på spørsmål 1 – kan test da benyttes som diagnoseverktøy for å påvise infeksjon av SARS-CoV-2?

3. Er PCR-testen validert på molekylnivå?

4. Er det i testen kontroll for positivt å påvise SARS-CoV-2 eller negativt å utelukke tilstedeværelsen av andre koronavirus?

5. Finnes det en Standard Operational Protocol til dette?

6. Hvis nei på disse spørsmål – er testet egnet som diagnoseverktøy for SARS-CoV-2?

E. Nedstenging har ingen virkelig betydning, heter det i denne studien:

https://www.derimot.no/ny-undersokelse-fra-standford-universitetet-ingen-forskjell-i-effekten-av-harde-og-milde-covid-19-tiltak/

Spørsmål:

1. Kjenner dere til denne studien?

2. Har nedstengingen virkelig en betydning slik dere ser det?

3. Hvordan vet vi vitenskapelig at nedstengning har en positiv ønsket betydning?

F. Spørsmål omkring Nordre Follo og de engelske muterte utgaven av SARS-Cov-2. I VG 22.1. står det at en ny metodikk «ble etablert for bare noen dager siden». Det het også «Mange helgenomsekvenseres».

Spørsmål:

1. Kan dere konkretisere hva slags metodikk det konkret er – og hvor den kommer fra?

2. Hva betyr helgenomsekvenseres – hva er det?

3. Er det forsvarlig å stenge ned næringsliv og kjøpesenter basert på en ny metodikk?

4. Har utenforstående dobbeltsjekket den nye metodikken?

5. Hva er det vitenskapelig grunnlaget for den?

6. Vil dere omgående sende oss den vitenskapelige dokumentasjon som bekrefter funn av dette muterte Sars Cov2-virus?

7. Vil dere omgående sende oss informasjon om metoden dere har benyttet for å bekrefte eksistensen av dette virus?

G. NRK hadde mandag 25.1 en sak: «Grafikken viser mutasjonen 501, som har gitt navn både til den britiske og den sørafrikanske mutasjonen av koronaviruset. Utbruddet i Nordre Follo skyldes den britiske (engelske) varianten.» https://www.nrk.no/norge/slik-skal-fhi-jakte-den-muterte-virusvarianten_-tre-grep-blir-viktige-1.15343389

Spørsmål:

1. På hvilken måte er grafikken representativ for mutasjonen?

2. Hvem har laget grafikken?

3. Har dere et bilde av viruset med mutasjonen slik at vi kan vurdere det med lekfolks blikk?

4. Hvordan vil dere påvist viruset framover i lys at den nye informasjonen fra WHO?

5. Lederen av Folkehelseinstituttet og helseministeren virker å være uenig om det er mulig å bli kvitt det muterte viruset – hva er grunnen?

6. Det finnes visstnok tusenvis av muterte utgaver av SARS-CoV-2, har man oversikt over disse og hvorfor er den engelske utgaven spesielt farlig?

H. Slik vi oppfatter situasjonen har regjeringen via dekreter og en spesiell tolkning av smittevernloven satt til side andre lover som Grunnloven, arbeidsmiljøloven, barnevernsloven og menneskerettighetskonvensjoner på grunn av korona-pandemien.

Spørsmål:

1. Tilfredsstiller PCR-prøven med sine enormt mange positive utslag kravene i smittevernloven?

2. Er regjeringen gått for langt i sine tiltak, særlig med tanke på den nye informasjonen fra WHO?

3. Har regjeringens handling vært ulovlige?

4. Vil styresmaktene i praksis følge opp denne informasjonen fra WHO og legge om sin smittevernpolitikk?

5. Vil kommuner, helseforetak og andre relevante få be beskjed innen kort tid?

6. Kommunen har et selvstendig ansvar for sine tiltak – kan dere bekrefte dette?

7. Folkehelseinstituttet er registrert som FOLKEHELSEINSTITUTTET i Brønnøysundregistrene, org nr 983744516, har dette noen form for betydning for instituttets forståelse og tolkninger av Grunnloven og andre relevant lover og regler i Norge?

Med vennlig hilsen innbyggere i Vestfold og Telemark

Kontakt: innbyggerhelse@protonmail.com


Vedlegg og informasjon:

Link til informasjonen fra WHO: https://www.who.int/news/item/20-01-2021-who-information-notice-for-ivd-users-2020-05

WHO Information Notice for IVD Users 2020/05

Nucleic acid testing (NAT) technologies that use polymerase chain reaction (PCR) for detection of SARS-CoV-2

20 January 2021

Medical product alert

Geneva

Reading time: 1 min (370 words)

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Product type: Nucleic acid testing (NAT) technologies that use polymerase chain reaction (PCR) for detection of SARS-CoV-2

Date: 13 January 2021

WHO-identifier: 2020/5, version 2

Target audience: laboratory professionals and users of IVDs.

Purpose of this notice: clarify information previously provided by WHO. This notice supersedes WHO Information Notice for In Vitro Diagnostic Medical Device (IVD) Users 2020/05 version

1, issued 14 December 2020.

Description of the problem: WHO requests users to follow the instructions for use (IFU) when interpreting results for specimens tested using PCR methodology.

Users of IVDs must read and follow the IFU carefully to determine if manual adjustment of the PCR positivity threshold is recommended by the manufacturer.

WHO guidance Diagnostic testing for SARS-CoV-2 states that careful interpretation of weak positive results is needed (1). The cycle threshold (Ct) needed to detect virus is inversely proportional to the patient’s viral load. Where test results do not correspond with the clinical presentation, a new specimen should be taken and retested using the same or different NAT technology.

WHO reminds IVD users that disease prevalence alters the predictive value of test results; as disease prevalence decreases, the risk of false positive increases (2). This means that the probability that a person who has a positive result (SARS-CoV-2 detected) is truly infected with SARS-CoV-2 decreases as prevalence decreases, irrespective of the claimed specificity.

Most PCR assays are indicated as an aid for diagnosis, therefore, health care providers must consider any result in combination with timing of sampling, specimen type, assay specifics, clinical observations, patient history, confirmed status of any contacts, and epidemiological information.

Actions to be taken by IVD users:

1. Please read carefully the IFU in its entirety.

2. Contact your local representative if there is any aspect of the IFU that is unclear to you.

3. Check the IFU for each incoming consignment to detect any changes to the IFU.

4. Provide the Ct value in the report to the requesting health care provider.

Contact person for further information:

Anita SANDS, Regulation and Prequalification, World Health Organization, e-mail: rapidalert@who.int

References:

1. Diagnostic testing for SARS-CoV-2. Geneva: World Health Organization; 2020, WHO reference number WHO/2019-nCoV/laboratory/2020.6.

2. Altman DG, Bland JM. Diagnostic tests 2: Predictive values. BMJ. 1994 Jul 9;309(6947):102. doi: 10.1136/bmj.309.6947.102.

FRA SELVE WHO-DOKUMENT SEPEMBER 2020:

OGSÅ HER KOMMER DET FRAM UTFORDRINGER KNYTTET TIL PCR-TESTEN

Respiratory secretions may be quite variable in composition, and the adequacy of sampling efforts may also vary, which can occasionally result in false-negative PCR results [40, 42, 58, 66-74]. In patients for whom SARS-CoV-2 infection is strongly suspected and URT swabs are negative, viral RNA may be detected in LRT secretions, such as sputum or bronchoalveolar lavage [70, 71, 75, 76]. Faeces or rectal swabs have been shown to be positive for SARS-CoV-2 RNA in a subset of patients, with some studies suggesting that this positivity is prolonged compared to that of respiratory tract specimens [46, 56, 59, 75, 77]. In some patients, SARS-CoV-2 RNA detection in blood samples has been reported and some studies suggest that detection in the blood is associated with disease severity, however, more studies on this potential association are required [75, 78-81]. In oral fluid specimens (e.g. induced saliva) [28, 49, 82-88], reported detection rates compared with URT specimens from the same patient vary widely, and limited data are available on adequacy of SARS-CoV-2 detection in gargling/mouth washes [85]. The striking differences in sensitivity of oral fluids evaluations are potentially due to large differences in collection, transport and storage techniques, as well as the evaluation of different testing populations.

Individuals infected with SARS-CoV-2 may never develop symptoms (asymptomatic cases), they may have very mild disease (pauci-symptomatic), or they may develop moderate to severe COVID-19 disease [18-26]. The most robust evidence for viral infection comes from the detection of fragments of the virus, such as proteins or nucleic acids, through virological testing.

Infected individuals may test positive for viral nucleic acids or viral proteins without symptoms (asymptomatic), or before symptom onset (pre-symptomatic), and throughout a disease episode (symptomatic). For those who develop COVID-19 illness, symptoms can be wide-ranging at initial presentation of disease. Individuals may present with very mild symptoms, with apparent pneumonia, febrile illnesses/sepsis, and less commonly with gastro-enteritis or neurological symptoms [99]. If required for case management, patients should also be tested for other pathogens, as recommended in local clinical management guidelines, but this should never delay testing for SARS-CoV-2 [99, 100]. Co-infections of SARS-CoV-2 with other pathogens have been reported, thus a positive test for another pathogen does not rule out COVID-19 and vice versa [27, 101-109]. Cases of false positive dengue antibody test results using a dengue rapid diagnostic test (RDT) in COVID-19 patients have been reported [110, 111]. There is also a risk of false positive or false negative SARS-CoV-2 results, if testing is not performed with adequate assays or not done under adequate conditions.

Biosafety practices in the laboratory

Laboratories undertaking testing for SARS-CoV-2 should adhere strictly to the appropriate biosafety practices. Testing clinical specimens that may contain SARS-CoV-2 should be performed in appropriately equipped laboratories by staff trained in the relevant technical and safety procedures. National guidelines on laboratory biosafety should be followed in all circumstances.

Specimen handling for molecular testing using standard rRT-PCR requires biosafety level (BSL) 2 or equivalent facilities with the use of a biosafety cabinet (BSC) or a primary containment device that is recommended for sample manipulation before inactivation.Attempts to isolate the virus in cell culture require BSL-3 facilities at a minimum. When performing viral culture on potentially SARS-CoV-2 positive clinical specimens for other purposes, a risk assessment needs to be conducted followed by required safety measures and procedures [136].

Specific considerations of biosafety requirements may allow certain point-of-care (POC) or near-patient assays to be performed outside a biosafety cabinet, once local regulations have been reviewed, after performing risk assessment and having put in place adequate risk-mitigation measures. For further details on laboratory biosafety, see specific laboratory biosafety interim guidance [3]. For general laboratory biosafety guidelines, see the WHO Laboratory biosafety manual, 3rd edition [136].’

Testing for SARS-CoV-2

Nucleic acid amplification test (NAAT)

Wherever possible, suspected active SARS-CoV-2 infections should be tested with NAAT, such as rRT-PCR. NAAT assays should target the SARS-CoV-2 genome. Since there is currently no known circulation of SARS-CoV-1 globally, a sarbecovirus-specific sequence is also a reasonable target. For commercial assays, interpretation of results should be done according to the instructions for use. Optimal diagnostics consist of a NAAT assay with at least two independent targets on the SARS-CoV-2 genome, however, in areas with widespread transmission of SARS-CoV-2, a simple algorithm might be adopted with one single discriminatory target. When using a one-target assay, it is recommended to have a strategy in place to monitor for mutations that might affect performance. For more details, see section below on “Background information on monitoring for mutations in primer and probe regions”.’

Background information on monitoring for mutations in primer and probe regions

As SARS-CoV-2 continues to acquire genetic changes over time, mismatches between primers and/or probes, and corresponding binding sites within SARS-CoV-2 genomes may reduce NAAT sensitivity. Where feasible, monitor for primer and probe mismatches due to SARS-CoV-2 mutations, and assess their impact. By routinely testing all specimens with two different primer/probe sets that target different genomic regions it is possible to reduce the risk of false-negative results. Several tools monitoring for relevant mutations are available, including searches done by GISAID (the Global Initiative on Sharing All Influenza Data) and other tools including PrimerCheck (Erasmus Medical Centre), PrimerScan (European Centre for Disease prevention and Control) and CoV-GLUE (COVID-19 UK Genomics Consortium and MRC-University of Glasgow Centre for Virus Research). Primercheck and COV-GLUE allows researchers to use their own sequence data confidentially as input. Not all mutations in primer/probe regions lead to significant changes in performance. In silico predictions of binding efficiency are insufficient to quantify the effect of a mismatch on the sensitivity of a NAAT, so it is essential to do an experimental comparison of the assay’s sensitivity for both variant and reference virus isolates. For commercial assays, it is vital to keep track of possible incidents of suboptimal performance. Inform the manufacturer of the assay and WHO of any concerns you may experience with a specific assay.

Such systems provide access to testing in locations with limited laboratory capacity and rapid turnaround time when used for near-patient testing. Validation data of some of these assays are now available [144]. When implementing these assays in specific settings, staff performing the test should be appropriately trained, performance should be assessed in those specific settings and a system to monitor quality should be put in place. Additional potentially valuable amplification/detection methods, such as CRISPR (targeting clustered regularly interspaced short palindromic repeats), isothermal nucleic acid amplification technologies (e.g. reverse transcription loop-mediated isothermal amplification (RT-LAMP), and molecular microarray assays are under development or in the process of being commercialized [145-147]. Validation of the analytic and clinical performance of these assays, demonstration of their potential operational utility, rapid sharing of data, as well as emergency regulatory review of manufacturable, well-performing tests are encouraged to increase access to SARS-CoV-2 testing.

Careful interpretation of weak positive NAAT results is needed, as some of the assays have shown to produce false signals at high Ct values. When test results turn out to be invalid or questionable, the patient should be resampled and retested. If additional samples from the patient are not available, RNA should be re-extracted from the original samples and retested by highly experienced staff. Results can be confirmed by an alternative NAAT test or via virus sequencing if the viral load is sufficiently high. Laboratories are urged to seek reference laboratory confirmation of any unexpected results.

One or more negative results do not necessarily rule out the SARS-CoV-2 infection [40, 42, 58, 66-74]. A number of factors could lead to a negative result in an infected individual, including:

– poor quality of the specimen, because it contains too little patient material;

– the specimen was collected late in the course of the disease, or the specimen was taken from a body compartment that did not contain the virus at that given time;

– the specimen was not handled and/or shipped appropriately;

– technical reasons inherent in the test, e.g. PCR inhibition or virus mutation.

Alternatives to RNA extraction

Most conventional molecular diagnostic workflows require RNA extraction before an rRT-PCR test is conducted. However, there are global shortages of commercial extraction kits due to the COVID-19 pandemic. Direct rRT-PCR from nasopharyngeal swabs may provide an emergency or temporary alternative to RNA extraction, but limitations to the input volume, as well as an increased risk of RNA degradation and PCR inhibition can lead to a loss of sensitivity of the assay [148, 149]. Heat treatment prior to sample processing can affect the RNA quality [149, 150]. Other factors that can affect RNA quality and which should be evaluated before implementation are the addition of detergents, transport media, the volume of the specimen used, and the polymerase enzyme used [148, 151-154]. The biosafety implications of alternative extraction workflows should also be considered. Laboratories considering alternative methods that bypass the need for RNA extraction should validate their protocols thoroughly and conduct a risk assessment that weighs the benefits and risks, before integrating such protocols into a diagnostic workflow.

Pooling of specimens for NAAT << the act of sharing or combining two or more things:

Pooling of samples from multiple individuals can increase the diagnostic capacity for detecting SARS-CoV-2 when the rate of testing does not meet the demand in some settings [155-159].

There are several strategies for pooling specimens. If the pooled result is negative, all individual specimens in the pool are regarded as negative. If the pool tests positive the follow-up steps depend on the strategy, but in general each specimen needs to undergo individual testing (pool deconvolution) to identify the positive specimen(s). Another approach is matrix pooling. This means that pools are made per row and per column, and tested by PCR, the position in the matrix identifies the positive specimen without additional testing if prevalence is sufficiently low.

Depending how robust the matrix testing method is in the specific context, it might still be advisable to retest the identified positive samples for confirmation. Pooling of specimens could be considered in population groups with a low/very low expected prevalence of SARS-CoV-2 infection, but not for cases or cohorts that more likely to be infected with SARS-CoV-2. Routine use of the pooling of specimens from multiple individuals in clinical care and for contact tracing purposes is not recommended. Studies have been conducted to determine the optimal sample pooling number and design pooling strategies in different outbreak settings [156, 160-162].

Before any sample pooling protocols can be implemented, they must be validated in the appropriate populations and settings. An inappropriate testing strategy may lead to missed cases or other laboratory errors that may, in turn, negatively affect patient management and public health control measures. In addition, the risk of cross-contamination and the potential increase in workload complexity and volume must be considered. To perform reliable pooling, adequate automation is key (e.g. robotic systems, software supporting the algorithms to identify positive samples, laboratory information systems and middle-ware that can work with sample pooling).

Based on currently available data, intra-individual pooling (multiple specimens from one individual that are pooled and tested as a single sample) from upper respiratory tract samples can be used. Intra-individual pooling of sputum and faeces with URT samples is not recommended because the former may contain compounds that inhibit rRT-PCR.

Rapid diagnostic tests based on antigen detection

Rapid diagnostic tests that detect the presence of SARS-CoV-2 viral proteins (antigens) in respiratory tract specimens are being developed and commercialized. Most of these are lateral flow immunoassays (LFI), which are typically completed within 30 minutes. In contrast to NAATs, there is no amplification of the target that is detected, making antigen tests less sensitive.

Additionally, false-positive (indicating that a person is infected when they are not) results may occur if the antibodies on the test strip also recognize antigens of viruses other than SARS-CoV-2, such other human coronaviruses.

The sensitivity of different RDTs compared to rRT-PCR in specimens from URT (nasopharyngeal swabs) appears to be highly variable [144, 163-165], but specificity is consistently reported to be high. Currently, data on antigen performance in the clinical setting is still limited: paired NAAT and antigen validations in clinical studies are encouraged to identify which of the antigen detection tests that are either under development or have already been commercialized demonstrate acceptable performance in representative field studies. When performance is acceptable, antigen RDTs could be implemented in a diagnostic algorithm to reduce the number of molecular tests that need to be performed and to support rapid identification and management of COVID-19 cases. How antigen detection would be incorporated into the testing algorithm depends on the sensitivity and specificity of the antigen test and on the prevalence of SARS-CoV-2

infection in the intended testing population. Higher viral loads are associated with improved antigen test performance; therefore test performance is expected to be best around symptom onset and in the initial phase of a SARS-CoV-2 infection. For specific guidance on antigen detection tests see the WHO Interim guidance on antigen-detection in diagnosis of SARS-CoV-2 infection using rapid immunoassays [5].

Antibody testing < a protein produced in the blood that fights diseases by attacking and killing harmful bacteria, viruses, etc.:

Serological assays that detect antibodies produced by the human body in response to infection with the SARS-CoV-2 can be useful in various settings.

For example, serosurveillance studies can be used to support the investigation of an ongoing outbreak and to support the retrospective assessment of the attack rate or the size of an outbreak [9]. As SARS-CoV-2 is a novel pathogen, our understanding of the antibody responses it engenders is still emerging and therefore antibody detection tests should be used with caution, and not used to determine acute infections.

Non-quantitative assays (e.g. lateral flow assays) cannot detect an increase in antibody titres, in contrast to (semi)quantitative or quantitative assays. Lateral flow antibody detection assays (or other non-quantitative assays) are currently not recommended for acute diagnosis and clinical management and their role in epidemiologic surveys is being studied. For more information on the utility of rapid immunodiagnostic tests, we refer to the WHO scientific brief with advice on the specific SARS-CoV-2 point-of-care immunodiagnostic tests [4].

Serology should not be used as a standalone diagnostic to identify acute cases in clinical care or for contact tracing purposes. Interpretations should be made by an expert and are dependent on several factors including the timing of the disease, clinical morbidity, the epidemiology and prevalence within the setting, the type of test used, the validation method, and the reliability of the results.

Seroconversion (development of measurable antibody response after infection) has been observed to be more robust and faster in patients with severe disease compared to those with milder disease or asymptomatic infections. Antibodies have been detected as early as in the end of the first week of illness in a fraction of patients, but can also take weeks to develop in patients with subclinical/mild infection [37, 166-173]. A reliable diagnosis of COVID-19 infection based on patients’ antibody response will often only be possible in the recovery phase, when opportunities for clinical intervention or interruption of disease transmission have passed. Therefore, serology is not a suitable replacement for virological assays to inform contact tracing or clinical management. The duration of the persistence of antibodies generated in response to SARS-CoV-2 is still under study [49, 174]. Furthermore, the presence of antibodies that bind to

SARS-CoV-2 does not guarantee that they are neutralizing antibodies, or that they offer protective immunity.

Available serological tests for detecting antibodies

Commercial and non-commercial tests measuring binding antibodies (Total immunoglobulins (Ig), IgG, IgM, and/or IgA in different combinations) utilizing various techniques including LFI, enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLIA) have become available. A number of validations and systematic reviews on these assays have been published [170, 171, 173, 175-177]. The performance of serologic assays varies widely in different testing groups (such as in patients with mild versus moderate-to-severe disease as well as in young versus old), timing of testing and the target viral protein. Understanding these performance variations will require further study. Antibody detection tests for coronavirus may also cross-react with other pathogens, including other human coronaviruses, [167, 178-180] or with pre-existing conditions (e.g. pregnancy, autoimmune diseases) and thus yield false-positive results.

Virus neutralization assays are considered to be the gold standard test for detecting the presence of functional antibodies. These tests require highly skilled staff and BSL-3 culture facilities and, therefore, are unsuitable for use in routine diagnostic testing.

Implementation and interpretation of antibody testing in the clinical laboratory

When implementing serological assays in the clinical laboratory, an in-house validation or verification of the specific assays is advisable. Even if commercial tests have been authorized for use in emergencies, an in-house verification (or if required by local authorities a validation) is still required. Protocols and examples with suggestions as to how to do this are now available [170, 171, 181].

Each serological test is different. With regard to commercial tests, follow the manufacturer’s instructions for use. Studies show that several commercial assays measuring total Ig or IgG have performed well. Most of these studies show no advantage of IgM over IgG, as IgM does not appear much earlier than IgG [173]. The additional role of IgA testing in routine diagnostics has not been established. For confirmation of a recent infection, acute and convalescent sera must be tested using a validated (semi)quantitative or quantitative assay. The first sample should be collected during the acute phase of illness, and the second sample at least 14 days after the initial sera was collected. Maximum antibody levels are expected to occur in the third/fourth week after symptom onset. Seroconversion or a rise in antibody titres in paired sera will help to confirm whether the infection is recent and/or acute. If the initial sample tests positive, this result could be due to a past infection that is not related to the current illness.

The first known case of reinfection with SARS-CoV-2 has been documented [182]. Only limited information is available on the interpretation of SARS-CoV-2 antibody tests after a previous infection with SARS-CoV-2 and on the dynamics of SARS-CoV-2 serology if a subsequent infection with another coronavirus occurs. In these two sets of circumstances interpretation of serology may be extremely challenging.

Viral isolation

Virus isolation is not recommended as a routine diagnostic procedure. All procedures involving viral isolation in cell culture require trained staff and BSL-3 facilities. A thorough risk assessment should be carried out when culturing specimens from potential SARS-CoV-2 patients for other respiratory viruses because SARS-CoV-2 has been shown to grow on a variety of

cell lines [183].

Genomic sequencing for SARS-CoV-2 relating to the complete set of genetic material of a human, animal, plant, or other living thing: Genomic sequencing for SARS-CoV-2 can be used to investigate the dynamics of the outbreak, including changes in the size of an epidemic over time, its spatiotemporal spread, and testing hypotheses about transmission routes. In addition, genomic sequences can be used to decide which diagnostic assays, drugs and vaccines may be suitable candidates for further exploration.

Analysis of SARS-CoV-2 virus genomes can, therefore, complement, augment and support strategies to reduce the disease burden of COVID-19. However, the potentially high cost and volume of the work required for genomic sequencing means that laboratories should have clarity about the expected returns from such investment and what is required to maximize the utility of such genomic sequence data. WHO guidance on SARS-CoV-2 genomic sequencing is currently being developed.

Quality assurance

Before introducing a new testing method, a new assay, new batches of materials, or a new PCR technician into the laboratory, a validation or verification should be carried out, to ensure that the laboratory testing system is performing adequately.

For manual PCR systems, each NAAT sample should include internal controls and ideally a specimen collection control (human gene target). Additionally, external controls are recommended for each test run. Laboratories that order their own primers and probes should carry out entry testing or validation looking at functionality and potential contaminants [184].

Laboratories are encouraged to define their assays’ detection limits, and senior staff should recognize how disease prevalence alters the predictive value of their test results. Once the number of cases goes down, the positive predictive value will decrease, therefore the interpretation of tests should continue to be part of a stringent quality assurance scheme, with interpretation based on: timing of sampling, sample type, test specifics, clinical data and epidemiological data.

Laboratories should put measures in place to reduce the potential for false positive rRT-PCR results and have a strategy for the management of equivocal results. See Annex 4 for a checklist.

In general, laboratories should have a quality assurance system in place and are encouraged to participate in external quality assessment (EQA) schemes or perform result comparison between laboratories of a subset of samples.

WHO has previously advised national laboratories to ensure quality performance by confirmation of testing results for the first 5 positive specimens and the first 10 negative specimens (collected from patients that fit the case definition) by referring them to one of the WHO reference laboratories that provide confirmatory testing for SARS-CoV-2. WHO provided support to national laboratories to facilitate specimen shipment to one of the dedicated reference laboratories. For more information, consult the WHO website for the list of reference laboratories [185] and shipment instructions [135]. Strengthened national reference laboratories and growing access to EQA schemes for SARS-CoV-2 reduce the need to use this mechanism. If testing for SARS-CoV-2 is not yet available in a country, efforts should be made to establish national capacity.

Reporting of cases and test results

Rapid communication of test results is important for planning and design of public health and outbreak control interventions. Laboratories should follow national reporting requirements. In general, all test results, positive or negative, should be immediately reported to the national authorities. States Parties to the International Health Regulations (IHR) are reminded of their obligation to share with WHO relevant public health information for events for which they must notify WHO, using the decision instrument in Annex 2 of the IHR (2005) [186].

Regular interaction between public health experts, clinicians and local laboratory experts to discuss strategies, potential problems and solutions, should be considered to be an essential part of an adequate COVID-19 response. This response includes the development of guidance and (clinical-, epidemiological-, and trial) study protocols.

A rapid turnaround time of test results can, in turn, have a positive impact on the outbreak [187, 188]. More studies are needed to fine tune the maximum acceptable time from symptom onset to sample result to have impact on clinical management and outbreak control; currently a maximum of 24 hours is considered reasonable in most settings. As laboratories often have control only over the time between sample arrival and the test result, it is critical to ensure that samples arrive in the laboratory without delay.

This is the sixth edition (version 2020.6) and was originally adapted from Laboratory testing for Middle East Respiratory Syndrome Coronavirus [189].A broad spectrum of clinical laboratory experts from different regions were engaged in the development of this document. The internal experts involved in the development include WHO regional laboratory focal points, epidemiologists and clinical experts. This version of the guidance incorporates the novel understanding and characteristics of the virus and addresses questions and issues received from WHO’s country and regional offices and other channels.

Contributors

WHO steering group: Amal Barakat, Céline Barnadas, Silvia Bertagnolio, Caroline Brown, Lisa Carter, Sebastian Cognat, Jane Cunningham, Varja Grabovac, Francis Inbanathan, Kazunobu Kojima, Juliana Leite, Marco Marklewitz, Jairo Mendez-Rico, Karen Nahapetyan, Chris Oxenford, Boris Pavlin, Mark Perkins, Anne Perrocheau, Jose Rovira, Maria Van Kerkhove, Karin von Eije, Joanna Zwetyenga,

External contributors:

Sarah Hill, Oxford University and Royal Veterinary College, United Kingdom; Maria Zambon, Public Health England, United Kingdom; Corine Geurts van Kessel, Richard Molenkamp and Marion Koopmans, Erasmus MC and Adam Meijer and Chantal Reusken, RIVM, The Netherlands; Antonino Di Caro, Istituto Nazionale per le Malattie Infettive Lazzaro Spallanzani, Italy; Anne von Gottberg, National Institute for Communicable Diseases, South Africa; Janejai Noppavan, National institute of Health, Thailand; Raymond Lin, National Public Health Laboratory,

Singapore; Leo Poon and Malik Peiris, Hong Kong University, China, Hong Kong SAR; George Gao, Chinese CDC, China.

References

1. Laboratory testing strategy recommendations for COVID-19. World Health Organization 2020; Available from: https://apps.who.int/iris/handle/10665/331509.

2. Laboratory assessment tool for laboratories implementing COVID-19 virus testing. World Health Organization 2020 8 April 2020 7 July 2020]; Available from:

https://apps.who.int/iris/handle/10665/331715.

3. Laboratory biosafety guidance related to coronavirus disease (COVID-19). World Health Organization 2020; Available from: https://apps.who.int/iris/handle/10665/332076

4. Advice on the use of point-of-care immunodiagnostic tests for COVID-19. World Health Organization 8 April 2020; Available from: https://apps.who.int/iris/handle/10665/331713.

5. Antigen detection in diagnosis of SARS-CoV-2 infection using rapid immunoassays, interim guidance. World Health Organization 2020. Available from:

https://apps.who.int/iris/handle/10665/334253

31. Li, Q., et al., Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N Engl J Med, 2020. 382(13): p. 1199-1207.

44. Hu, Z., et al., Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing, China. Sci China Life Sci, 2020. 63(5): p. 706-711.

45. Liu, Y., et al., Viral dynamics in mild and severe cases of COVID-19. Lancet Infect Dis, 2020.

46. Zheng, S., et al., Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January-March 2020: retrospective cohort study. BMJ, 2020. 369: p. m1443.

Chen, X., et al., Detectable serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients. Clin Infect Dis, 2020.

80. Corman, V.M., et al., SARS-CoV-2 asymptomatic and symptomatic patients and risk for transfusion

125. Chen L, Z.J., Peng J, Li X, Deng X, Shen Z, Guo F, Zhang Q, Zhang Q, Jin Y, Wang L, Wang S Detection of 2019-nCoV in Saliva and Characterization of Oral Symptoms in COVID-19 Patients. SSRN, 2020.

126. Ng, S.C., F.K.L. Chan, and P.K.S. Chan, Screening FMT donors during the COVID-19 pandemic: a protocol for stool SARS-CoV-2 viral quantification. Lancet Gastroenterol Hepatol, 2020.5(7): p. 642-643.

127. Tang, J.W., et al., Quantitative temporal-spatial distribution of severe acute respiratory syndrome-

Many kits require cold chain conditions during shipment and storage, in some circumstances this might pose a challenge. Some kits contain lyophilized enzymes that do not require the kit to be shipped and sometimes stored cold.

Shelf life: To be prepared for periods of intense testing stocks might be needed, a longer shelf life is needed to ensure adequate use of resources.

Complete kit for sampling/extraction/amplification or the PCR kit requires additional reagents or tools.

Compatibility with laboratories’ extraction method.

Compatibility with procurable polymerases if needed.

Special equipment needed (e.g. calibration panel before running the test, extraction platforms, heat block, vortex, magnetic stand or centrifuge).

Laboratories should have a standard operating procedure in place to reduce the possible false positive rRT-PCR results and how to handle equivocal results. This checklist provides the laboratories with suggestions and considerations. The checklist is formulated for manual rRT-PCRs but many aspects can also be used for other NAATs.

CLERICAL

Eliminate or reduce transcription

If transcribe, method of checking

Sorting, aliquoting and labelling

Double identifiers

Entering results

CROSS CONTAMINATION

Preparation area

Manipulation of tubes

EQUIPMENT and TEST KITS

Calibration method

Equipment validated for test kit

Assess new equipment for contamination risk

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