ANNEX XIII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Table 1 applies to the following first-line assays (including rapid tests) for antibodies against SARS-CoV-2 (anti-SARS-CoV-2): total antibody, IgG-only, IgG combined with IgM and/or IgA.

Table 2 applies to first-line assays (including rapid tests) for detection of anti-SARS-CoV-2 IgM and/or IgA.

Table 3 applies to confirmatory or supplemental assays for anti-SARS-CoV-2.

Table 4 applies to antigen SARS-CoV-2 tests, including rapid antigen tests.

Table 5 applies to NAT assays for SARS-CoV-2 RNA.

Table 6 applies to SARS-CoV-2 antigen self-tests which have already undergone a performance evaluation for professional use.

Table 7 applies to SARS-CoV-2 antibody self-tests which have already undergone a performance evaluation for professional use.

Table 1: First-line assays (including rapid tests) for anti-SARS-CoV-2: total antibody, IgG-only, IgG combined (1) with IgM and/or IgA

Performance characteristic Specimen Specimen number, features, use Acceptance criteria Diagnostic sensitivity Positive specimens ≥400 including specimens from early infection and post seroconversion (2) (within the first 21 days and after 21 days following the onset of symptoms); including specimens from asymptomatic or subclinical and mildly symptomatic (outpatient treatment) individuals; including specimens with low and high titers; including specimens from vaccinated individuals where appropriate (3); consideration of genetic variants ≥90% sensitivity (4) for specimens taken >21 days after onset of symptoms (5); overall sensitivity including the early infection phase shall be at least equivalent to the comparator device (6) Seroconversion panels As far as available Seroconversion sensitivity comparable to other CE-marked devices Analytical sensitivity Reference preparations WHO International Standard (IS) for anti- SARS-CoV-2 (NIBSC code 20/136); WHO International Reference Panel (RP) for anti-SARS-CoV-2 antibodies (NIBSC codes 20/140, 20/142, 20/144, 20/148, 20/150) IS: for titre determinations / quantitative (7) result output; RP: all antibody assays Diagnostic specificity Negative specimens (8) ≥400 specimens from non-infected and non-vaccinated individuals (9) >99% specificity (10) ≥200 hospitalised patients (without SARS-CoV-2 infection) Potential limitations for specificity, if any, shall beidentified Cross-reactivity Potentially cross-reacting specimens ≥100 in total including RF+, pregnant women, specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc.





Table 2: First-line assays (including rapid tests) for anti-SARS-CoV-2: IgM and/or IgA detection

Performance characteristic Specimen Specimen number, features, use Acceptance criteria Diagnostic sensitivity Positive specimens ≥200 (11) Specimens (12) with a significant proportion from the early phase of the infection (within 21 days after onset of symptoms) compared to specimens past seroconversion (>21 days after onset of symptoms); including specimens from asymptomatic, subclinical, mildly symptomatic (outpatient treatment) individuals; including freshly (13) vaccinated individuals if appropriate; consideration of genetic variants ≥80% sensitivity (14) for specimens taken during the first 21 days after symptom onset (15); overall sensitivity shall be at least equivalent to the comparator device (16) of the same type (i.e. IgM and/or IgA) Seroconversion panels As far as available Seroconversion sensitivity comparable to other CE-marked devices Analytical sensitivity Standards N/A N/A Diagnostic specificity Negative specimens (17) ≥200 specimens from non-infected and non-vaccinated individuals (18) ≥98% specificity (19) ≥100 from hospitalised patients (without SARS-CoV-2 infection) Potential limitations for specificity, if any, shall be identified Cross-reactivity Potentially cross-reacting specimens ≥100 in total including RF+, pregnant women, specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc.





Table 3: Confirmatory or supplemental (20) assays for anti-SARS-CoV-2

Performance characteristic Specimen Specimen number, features, use Acceptance criteria Diagnostic sensitivity Positive specimens ≥200 including specimens pre and post seroconversion (within the first 21 days and after 21 days following the onset of symptoms) Correct determination as “positive” (or “indeterminate”) Seroconversion panels/low titre panels as far as available Analytical sensitivity Standards N/A N/A Diagnostic specificity Negative specimens (21) ≥200 from non-infected / non-vaccinated population No false positive results; correct determination as “negative” (or “indeterminate”) ≥200 from hospitalised patients (without SARS-CoV-2 infection) Cross-reactivity Potentially cross-reacting specimens ≥50 in total including specimens with antibodies against endemic human coronaviruses 229E, OC43, NL63, HKU1 and other pathogens of respiratory diseases such as influenza A, B, RSV etc.; including specimens with indeterminate or false positive results in other anti-SARS-CoV-2 assays





Table 4: Antigen assays (including rapid tests): SARS-CoV-2

Performance characteristic Specimen Specimen number, features, use Acceptance criteria Diagnostic sensitivity Positive specimens ≥100 (22) NAT positive specimens (23) from early infection within the first 7 days after symptom onset (24); specimens shall represent naturally occurring viral loads (25); consideration of genetic variants (26); consideration of variations in specimen collection and/or specimen handling (27) Detection of >80% (rapid tests); detection of >85% (lab-based assays (28)); relative to SARS-CoV-2-NAT (29), (30) Analytical sensitivity Standards As soon as available Establishment of a LOD (31) Diagnostic specificity Negative specimens ≥300 from non-infected individuals Specificity >98% (rapid tests) Specificity >99% (lab-based assays (28)) ≥100 from hospitalised patients Potential limitations for specificity, if any, shall be identified Cross-reactivity Potentially cross-reacting specimens ≥50 in total including virus-positive specimens of endemic human coronaviruses 229E, OC43, NL63, HKU1; influenza A, B, RSV, and other pathogens of respiratory diseases, eligible for differential diagnosis; including bacteria (32) present in the specimen taking area





Table 5: NAT devices for SARS-CoV-2 RNA

Performance characteristic Specimen SARS-CoV-2 RNA qualitative SARS-CoV-2 RNA quantitative Sensitivity Analytical sensitivity: LOD WHO First International Standard SARS-CoV-2 RNA (NIBSC code 20/146; 7.70 Log10 IU/mL) Secondary standards calibrated against WHO IS According to Ph. Eur. NAT validation guideline: several dilution series into borderline concentration; statistical analysis (e.g. Probit analysis) on the basis of at least 24 replicates; calculation of 95 % cut-off value According to Ph. Eur. NAT validation guideline: several dilution series of calibrated reference preparations into borderline concentration; statistical analysis (e.g. Probit analysis) on the basis of at least 24 replicates; calculation of 95 % cut-off value as LOD Quantification limit; quantification features WHO First International Standard SARS-CoV-2 RNA (NIBSC code 20/146; 7.70 Log10 IU/mL) Secondary standards calibrated against WHO IS Dilutions (half-log10 or less) of calibrated reference preparations; determination of lower, upper quantification limit, LOD, precision, accuracy, “linear” measuring range, “dynamic range”. Synthetic target nucleic acid may be used as secondary standard to achieve higher concentration levels. Reproducibility at different concentration levels to be shown Diagnostic sensitivity: different SARS-CoV-2 RNA strains Patient specimens determined as SARS-CoV-2 RNA positive by comparator device from different regions and outbreak clusters; sequence variants Dilution series of SARS-CoV-2 positive cell cultures (isolates) may serve as potential substitutes ≥100 (33) Quantification efficiency SARS-CoV-2 RNA positive patient specimens from different regions and outbreak clusters; sequence variants with quantitative values obtained by comparator device Dilution series of SARS-CoV-2 RNA positive cell cultures may serve as potential substitutes ≥100 Inclusivity In silico analysis (34); at least two independent target gene regions in one test run (dual-target design) Evidence of suitable device design: primer/probe sequence alignments with published SARS-CoV-2 sequences Evidence of suitable device design: primer/probe sequence alignments with published SARS-CoV-2 sequences Specificity Diagnostic specificity SARS-CoV-2 RNA negative human specimens ≥500 ≥100 In silico analysis (34) Evidence of suitable device design (sequence alignments); regular check of primer/probe sequences against sequence data bank entries Evidence of suitable device design evidence (sequence alignments); regular check of primer/probe sequences against sequence data bank entries Cross-reactivity specimens positive (various concentrations) for related human coronaviruses 229E, HKU1, OC43, NL63, MERS coronavirus; SARS CoV-1 if available; Influenza virus A, B; RSV; Legionella pneumophila; positive cell cultures may serve as potential substitutes ≥20 in total ≥20 in total Robustness Carry-over At least 5 runs using alternating high positive and negative specimens. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. At least 5 runs using alternating high positive (known to occur naturally) and negative specimens Inhibition Internal control preferably to go through the whole NAT procedure Internal control preferably to go through the whole NAT procedure Whole system failure rate leading to false negative results: 99/100 assays positive ≥100 specimens virus-spiked with 3 × the 95 % positive cut-off concentration (3 x LOD) ≥100 specimens virus-spiked with 3 × the 95 % positive cut-off concentration (3 x LOD)





Table 6: Additional requirements for SARS-CoV-2 antigen self-tests (35)

Performance characteristic Specimens (36) Number of lay persons Result interpretation (37) Interpretation of results (38) by lay persons reflecting the following range of reactivity levels: — non-reactive — reactive — weak reactive (39) — invalid ≥100 Diagnostic sensitivity (40) Lay persons that are known antigen positive (41) , (42) ≥30 Diagnostic specificity (43) Lay persons that do not know their status (39) ≥60





Table 7: Additional requirements for SARS-CoV-2 antibody self-tests (44)

Performance characteristic Specimens (45) Number of lay persons Result interpretation (46) Interpretation of results (47) by lay persons reflecting the following range of reactivity levels: — non-reactive — reactive — weak reactive (48) — invalid ≥100 Diagnostic sensitivity (49) Lay persons that are known antibody positive (50) ≥100 Diagnostic specificity (51) Lay persons that do not know their status (48) ≥100

(1) Performance claim of the combined overall result; for devices with separate claims for IgM and/or IgA, see table 2.

(2) Details on the time interval between specimen taking and onset of symptoms (or time of infection, if available) shall be provided.

(3) The manufacturer shall provide a justification of the suitability and timing for sensitivity evaluation of the relevant antibodies in vaccinated individuals.

(4) Based on confirmed positive SARS-CoV-2 NAT result.

(5) Claims for sensitivity shall be specified in relation to the time between specimen taking after symptom onset or the initial PCR diagnosis and the test.

(6) CE marked under Regulation (EU) 2017/746 as class D, if available.

(7) This applies to quantitative assays if they are also first-line assays.

(8) Negative specimens shall be from individuals with no history of SARS-CoV-2 infection (if available pre-pandemic).

(9) Individuals vaccinated against an antigen different from that used in the device may be included, if appropriate.

(10) False positive results shall be resolved by re-testing in other SARS-CoV-2 serological assays, if necessary with different test design and antigen coating compared to the initial test, and/or confirmatory testing.

(11) In case of devices detecting both IgM and IgA, 200 per marker IgM and IgA.

(12) Details on the time interval between specimen taking and onset of symptoms (or time of infection, if available) shall be provided.

(13) The manufacturer shall provide a justification of the suitability and timing for sensitivity evaluation of IgM and IgA in vaccinated individuals.

(14) Diagnosis based on confirmed positive SARS-CoV-2 NAT result.

(15) Claims for sensitivity shall be specified in relation to the time between specimen taking after symptom onset or the initial PCR diagnosis and the test.

(16) CE marked under Regulation (EU) 2017/746 as class D, if available.

(17) Negative specimens shall be from individuals with no history of SARS-CoV-2 infection (if available pre-pandemic).

(18) Individuals vaccinated against an antigen different from that used in the device may be included, if appropriate.

(19) False positive results shall be resolved by re-testing in other SARS-CoV-2 serological assays, if necessary with different test design and antigen coating compared to the initial test, and/or confirmatory testing. Clarification of false positive results may additionally include testing for presence of other anti-SARS-CoV-2 antibody types (IgA, IgG, total antibody).

(20) E.g. immunoblot with antigens different from those used in the initial antibody test.

(21) Negative specimens shall be from individuals with no history of SARS-CoV-2 infection (if available pre-pandemic).

(22) If the device is intended to be used for more than one specimen type, 100 specimens shall be required for each specimen type. If this is not possible in exceptional circumstances (e.g. if specimen collection is very invasive), the manufacturer shall provide a justification and evidence of matrix equivalence.

(23) Specimen taking shall be matched for antigen and NAT testing, e.g., two simultaneous specimens from each individual or optimally NAT- and antigen testing from the same specimen (e.g. from the eluate of one swab); the buffer/transport medium shall be compatible with antigen testing; any volume change in the buffer/medium for specimen uptake between antigen and NAT device shall be clearly communicated.

(24) Or time of infection, if known, taking into account the incubation time.

(25) I.e., without preselection; the viral loads and their distribution shall be shown, e.g. characterized by Ct-values of RT-PCR; or transformed into viral load per ml of specimen, if applicable.

(26) Depending on the design of the device and nature of the genetic variant. For the purpose of evaluation, at least 3 specimens shall be represented for each relevant genetic variant.

(27) Specimen collection and extraction items such as swabs, extraction buffers, etc., shall be part of the evaluation. If proprietary specimen taking/preparation is not included in the device, device performance shall be investigated for an applicable range of specimen taking devices. If the specimen is not tested immediately, e.g. after a certain transport time, stability of the antigen shall be investigated.

(28) Other than rapid tests, i.e. formal laboratory-based devices e.g. enzyme immunoassay, automated tests, etc.

(29) The sensitivity of ≥80%, ≥85% respectively, shall be for all specimen types claimed. All claimed specimen types shall be compared with paired NAT results from nasopharyngeal specimens.

(30) The relationship between the sensitivity of the antigen test and of the NAT shall be demonstrated; sensitivity may be shown relating to different viral load ranges and to the threshold of infectivity. The NAT and extraction method used shall be described.

(31) Unless there is an available international standard, analytical sensitivity may be tested by dilution series of in-house virus preparations, comparatively with other antigen tests and NAT; if inactivated virus is used, the effect of inactivation and freeze/thawing on the antigen shall be investigated.

(32) E.g. staphylococci and streptococci expressing protein A or G.

(33) If the device is intended to be used for more than one specimen type, 100 specimens shall be required for each specimen type. If this is not possible in exceptional circumstances (e.g. if specimen collection is very invasive), the manufacturer shall provide a justification and evidence of matrix equivalence.

(34) The manufacturer shall document evidence of proactive regular surveillance checks against updated data bank entries in the post-market performance follow-up report.

(35) It is assumed that the underlying performance of the self-test has already been previously demonstrated with the evaluation/assessment of a professional test of the same design as the respective self-test under evaluation. In case for the self-use specimens in question there is no corresponding professional test variant, comparison shall be made with the standard specimen type (e.g. nasopharyngeal swabs for antigen test, serum or plasma for antibody test) of the corresponding professional test.

(36) For each self-use specimen type claimed with the device (e.g. nasal specimen, sputum, saliva, whole blood, etc.).

(37) The result interpretation study shall include reading and interpretation of the test results by at least 100 lay persons, with each lay person subjected to reading results covering the specified range of result reactivity levels. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(38) Tests shall be performed prior to the result interpretation study using whenever possible the specimen type intended by the manufacturer. Tests may be performed on contrived specimens based on the natural matrix of the respective specimen type.

(39) A higher proportion of the specimens shall be in the low-positive range close to the cut-off or LOD of the test.

(40) In comparison to RT-PCR. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(41) Individuals unaware of the professional diagnostic result prior to self-testing, and performing the entire test procedure from specimen collection and specimen pre-treatment (swab, buffer extraction, etc.) to reading.

(42) Subjects up to about 7 days after symptom onset.

(43) The manufacturer shall determine the concordance between lay person reading and professional user reading.

(44) It is assumed that the underlying performance of the self-test has already been previously demonstrated with the evaluation/assessment of a professional test of the same design as the respective self-test under evaluation. In case for the self-use specimens in question there is no corresponding professional test variant, comparison shall be made with the standard specimen type (e.g. nasopharyngeal swabs for antigen test, serum or plasma for antibody test) of the corresponding professional test.

(45) For each self-use specimen type claimed with the device (e.g. nasal specimen, sputum, saliva, whole blood, etc.).

(46) The result interpretation study shall include reading and interpretation of the test results by at least 100 lay persons, with each lay person subjected to reading results covering the specified range of result reactivity levels. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(47) Tests shall be performed prior to the result interpretation study using whenever possible the specimen type intended by the manufacturer. Tests may be performed on contrived specimens based on the natural matrix of the respective specimen type.

(48) A higher proportion of the specimens shall be in the low-positive range close to the cut-off or LOD of the test.

(49) With previous history of initial RT PCR-confirmed infection for SARS-CoV-2; in comparison to a previous confirmed antibody result. The manufacturer shall determine the concordance between lay person reading and professional user reading.

(50) Individuals unaware of the professional diagnostic result prior to self-testing, and performing the entire test procedure from specimen collection and specimen pre-treatment (swab, buffer extraction, etc.) to reading.

(51) The manufacturer shall determine the concordance between lay person reading and professional user reading.