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Commission Implementing Regulation (EU) 2022/1107 of 4 July 2022 laying down common specifications for certain class D in vitro diagnostic medical devices in accordance with Regulation (EU) 2017/746 of the European Parliament and of the Council (Text with EEA relevance)

Type Implementing Regulation

Publication 2022-07-05

State In force

Text As published

Department European Commission, SANTE

Source EUR-Lex

articles 4

This is the act as published. Later amendments are not folded into the text below: each one is a separate act in this repository and an entry in this law's history.

Reform history JSON API


Performance characteristic		anti-HBs	anti-HBc IgM	anti-HBe	HBeAg	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥100 vaccinees ≥100 naturally infected persons	≥200 Including specimens from different stages of infection (acute/chronic, etc.)	≥200 Including specimens from different stages of infection (acute/chronic, etc.)	≥200 Including specimens from different stages of infection (acute/chronic, etc.)	≥ 98 % (for anti-HBc IgM: applicable only on specimens from acute infection stage)
Diagnostic sensitivity	Seroconversion panels	10 anti-HBs seroconversion panels or follow-up series	When available	When available	When available	Diagnostic sensitivity during seroconversion shall represent the state of the art (for anti-HBc IgM, anti-HBe, HBeAg this shall be the case if applicable)
Analytical sensitivity	Standards	WHO Second International Standard for anti-hepatitis B surface antigen (anti-HBs) immunoglobulin, human NIBSC code: 07/164		WHO First International Standard anti-hepatitis B virus e antigen (anti-HBe), PEI code 129095/12	WHO First International Standard for Hepatitis B Virus e Antigen (HBeAg) PEI code 129097/12 HBe	anti-HBs: < 10 mIU/ml
Diagnostic specificity	Negative specimens	≥500 Including clinical specimens ≥50 potentially interfering specimens	≥200 blood donations ≥200 clinical specimens ≥50 potentially interfering specimens	≥200 blood donations ≥200 clinical specimens ≥50 potentially interfering specimens	≥200 blood donations ≥200 clinical specimens ≥50 potentially interfering specimens	≥ 98 %

Table 5. Qualitative and quantitative NAT devices for HBV DNA


	1.	For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection.

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	2.	Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens.

---	---	---
	3.	Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens.

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	4.	Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application. Performance characteristic Specimen Specimen numbers, features, use Acceptance criteria Analytical sensitivity WHO International Standard HBV DNA (or calibrated reference materials) NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device. LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit).²² quantitative NAT: definition of lower, upper quantification limit, precision, accuracy, ‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels According to the state of the art HBV genotype sensitivity WHO International Reference Panel HBV DNA (HBV genotypes) all relevant genotypes/subtypes, preferably from international reference materials potential substitutes for rare HBV genotypes (to be quantified by appropriate methods): plasmids; synthetic DNA Qualitative NAT: at least 10 specimens/genotype or subtype Quantitative NAT: dilution series for demonstration of quantification efficiencies According to the state of the art Diagnostic sensitivity Positive specimens reflecting the routine conditions of users (no pre-selection of specimens) Quantitative NAT: ≥100 Comparative results with another NAT system shall be generated in parallel According to the state of the art Seroconversion panels Qualitative NAT: ≥10 panels Comparative results with another NAT system shall be generated in parallel According to the state of the art Diagnostic specificity Blood donor specimens Qualitative NAT: ≥500 Quantitative NAT: ≥100 According to the state of the art Cross-reactivity Potentially cross-reacting specimens According to the state of the art Carry-over High HBV DNA positive; HBV DNA negative At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. According to the state of the art Detection in relation to antibody status HBV DNA positives: anti-HBV negative, anti-HBV positive Pre-seroconversion (anti-HBV negative) and post-seroconversion (anti-HBV positive) specimens According to the state of the art Whole system failure rate HBV DNA low-positive ≥100 HBV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. ≥99% positive

Table 6. Additional requirements for HBV self-tests

Performance characteristic	Specimens²³	Number of lay persons
Result interpretation²⁴	Interpretation of results²⁵ by lay persons reflecting the following range of reactivity levels: — non-reactive — reactive — weak reactive²⁶ — invalid	≥100
Diagnostic sensitivity	lay persons that are known positive	≥200
Diagnostic specificity	lay persons that do not know their status	≥400
Diagnostic specificity	lay persons that are at high risk of acquiring the infection	≥200

Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donor.

Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

For each body fluid claimed for use with the device, e.g. whole blood, urine, saliva, etc., the sensitivity and specificity of the device for self-testing in the hands of lay persons shall be defined against the confirmed patient infectious status.

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.

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

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

ANNEX VII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF HEPATITIS D VIRUS (HDV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of hepatitis D virus (HDV) infection.

Table 1 applies to devices intended for detection (including confirmation) or quantification of the following hepatitis D virus markers: antibodies against hepatitis D virus (anti-HDV), IgM antibodies against hepatitis D virus (anti-HDV IgM), the delta antigen.

Table 2 applies to qualitative and quantitative NAT devices for HDV RNA.

Table 1. Assays for HDV markers: anti-HDV, anti-HDV IgM, delta antigen


Performance characteristic		anti-HDV	anti-HDV IgM	Delta antigen	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥100 Specifying markers of HBV coinfection	≥50 Specifying markers of HBV coinfection	≥10 Specifying markers of HBV coinfection	≥ 98 %
Diagnostic specificity	Negative specimens	≥200 Including clinical specimens ≥50 potentially interfering specimens	≥200 Including clinical specimens ≥50 potentially interfering specimens	≥200 Including clinical specimens ≥50 potentially interfering specimens	≥ 98 %

Table 2. Qualitative and quantitative NAT devices for HDV RNA


	1.	For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection.

---	---	---
	2.	Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens.

---	---	---
	3.	Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens.

---	---	---
	4.	Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application. Performance characteristic Specimen Specimen numbers, features, use Acceptance criteria Analytical sensitivity WHO First International Standard HDV RNA, PEI code 7657/12 NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device. LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit).²⁷ quantitative NAT: definition of lower, upper quantification limit, precision, accuracy, ‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels According to the state of the art HDV genotype sensitivity all relevant genotypes/subtypes, preferably from international reference materials potential substitutes for rare HDV genotypes (to be quantified by appropriate methods): plasmids; synthetic RNA Quantitative NAT: dilution series for demonstration of quantification efficiencies According to the state of the art Diagnostic specificity Blood donor specimens Qualitative NAT: ≥100 Quantitative NAT: ≥100 According to the state of the art Cross-reactivity Potentially cross-reacting specimens According to the state of the art Carry-over High HDV RNA positive; HDV RNA negative At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. According to the state of the art Whole system failure rate HDV RNA low-positive ≥100 HDV RNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. ≥99% positive

Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

ANNEX VIII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OF MARKERS OF VARIANT CREUTZFELDT-JACOB (vCJD) DISEASE

Scope

This Annex applies to devices intended for detection of markers of variant Creutzfeldt-Jakob disease (vCJD).

Table 1 applies to devices intended for detection of markers of vCJD.

Table 1. Devices for detection of markers of vCJD

Performance characteristic	Material	Number of specimens	Acceptance criteria
Analytical sensitivity	vCJD brain spikes in human plasma (WHO reference number NHBY0/0003)	≥24 replicates of each of three dilutions of the material WHO number NHBY0/0003 (1×10⁴, 1×10⁵, 1×10⁶)	23 of the 24 replicates detected at 1×10⁴
Analytical sensitivity	vCJD spleen spikes in human plasma (10% spleen homogenate — NIBSC reference number NHSY0/0009)	≥24 replicates of each of three dilutions of the material NIBSC number NHSY0/0009 (1×10, 1×10² , 1×10³ )	23 of the 24 replicates detected at 1×10
Diagnostic sensitivity	Specimens from appropriate animal models	As many specimens as reasonably possible and available, and ≥10 specimens	90%
Diagnostic sensitivity	Specimens from humans with known clinical vCJD	As many specimens as reasonably possible and available, and ≥10 specimens	90%
Diagnostic sensitivity	Specimens from humans with known clinical vCJD	Only in cases where 10 specimens are not available: — the number of specimens tested shall be between 6 and 9 — all available specimens shall be tested	max. one false negative result
Analytical specificity	Potentially cross-reacting specimens	≥100
Diagnostic specificity	Normal human plasma specimens from area of low bovine spongiform encephalopathy (BSE) exposure	≥5 000	≥ 99,5 %

ANNEX IX

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF CYTOMEGALOVIRUS (CMV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of cytomegalovirus (CMV) infection.

Table 1 applies to first-line assays for total antibodies against CMV (total anti-CMV) and IgG antibodies against CMV (anti-CMV IgG).

Table 2 applies to qualitative and quantitative NAT devices for CMV DNA.

Table 1. First-line assays: total anti-CMV and anti-CMV IgG

Performance characteristic	Specimens	Specimen number, features, use	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥400 including specimens from recent and past CMV infection, low and high positive titre specimens	≥ 99% sensitivity for confirmable past infection²⁸; overall sensitivity including recent infection²⁹ shall be at least equivalent to the comparator device
Diagnostic sensitivity	Seroconversion panels	To be tested when available	Diagnostic sensitivity during seroconversion shall represent the state of the art
Analytical sensitivity	Standards	WHO International Standard anti-CMV IgG (PEI-code 136616/17) In case of titre determinations and quantitative statements
Diagnostic specificity	Negative specimens	≥400³⁰ CMV negative specimens from unselected donors, as compared to another CMV test.	≥ 99%
Diagnostic specificity	Hospitalised patients³¹	≥200	Potential limitations for specificity, if any, shall be identified
Cross-reactivity	Potentially cross-reacting³² specimens	≥100 in total (e.g. RF+, related viruses or other infectious agents, pregnant women, etc.)	Potential limitations for specificity, if any, shall be identified

Table 2. Qualitative and quantitative NAT devices for CMV DNA


	1.	For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection.

---	---	---
	2.	Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens.

---	---	---
	3.	Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens.

---	---	---
	4.	Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application. Performance characteristic Specimens Specimen numbers, features, use Acceptance criteria Analytical sensitivity WHO First International Standard Human CMV DNA (09/162; 5 000 000 IU/vial) (or calibrated reference materials) NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device. LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit).³³ quantitative NAT: definition of lower, upper quantification limit, precision, accuracy, ‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels According to the state of the art Diagnostic sensitivity CMV Strain sensitivity Patient specimens determined as CMV DNA positive by comparator device Dilution series of CMV positive cell cultures may serve as potential substitutes Qualitative NAT: ≥100 Quantitative NAT: ≥100 dilution series for demonstration of quantification efficiencies According to the state of the art Diagnostic specificity Blood donor specimens Qualitative NAT: ≥500 Quantitative NAT: ≥100 According to the state of the art Cross-reactivity Potentially cross-reacting specimens ≥20 specimens in total Including human specimens positive for related human herpesviruses, e.g. EBV, HHV6, VZV Herpesvirus positive cell cultures may serve as potential substitutes According to the state of the art Carry-over High CMV DNA positive; CMV DNA negative At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. According to the state of the art Whole system failure rate CMV DNA low-positive ≥100 CMV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. ≥99% positive

Including testing of other CMV parameters (e.g. CMV-IgM, avidity, immunoblot) or previous / follow-up specimens to assess true specimen status.

Supplementary testing to confirm recent CMV infection (primary or re-infection): e.g. CMV-IgM, IgG-avidity, immunoblot analysis.

Corresponding to an initial number of 1000 donors at an assumed CMV prevalence of 60 %.

Including pre-transplant recipients.

Including related β-herpes viruses (HHV-6, HHV-7).

Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

ANNEX X

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF EPSTEIN-BARR VIRUS (EBV) INFECTION

Scope

This Annex applies to devices intended for detection or quantification of markers of Epstein-Barr virus (EBV) infection.

Table 1 applies to first-line assays for IgG antibodies against viral capsid antigen of EBV (anti-EBV VCA IgG).

Table 2 applies to qualitative and quantitative NAT devices for EBV DNA.

Table 1: First-line assays: anti-EBV VCA IgG

Performance characteristic	Specimens	Specimen number, features, use	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥400 including specimens from recent and past EBV infection, low and high positive titre specimens	≥ 99% for confirmable past infection³⁴; overall sensitivity including recent infection³⁵ shall be at least equivalent to the comparator device
Diagnostic sensitivity	Seroconversion panels	To be tested when available	diagnostic sensitivity during seroconversion shall represent the state of the art
Analytical sensitivity	Standards	International reference reagents, when available
Diagnostic specificity	Negative specimens	≥ 200³⁶ EBV negatives from unselected donors as compared to another EBV device	≥ 99%
Diagnostic specificity	Hospitalised patients³⁷	≥200	Potential limitations for specificity, if any, shall be identified
Cross-reactivity	Potentially cross-reacting specimens	≥100 in total (e.g. RF+, related viruses or other infectious agents, pregnant women, etc.)	Potential limitations for specificity, if any, shall be identified

Table 2. Qualitative and quantitative NAT devices for EBV DNA


	1.	For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection.

---	---	---
	2.	Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens.

---	---	---
	3.	Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens.

---	---	---
	4.	Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application. Performance characteristic Specimens Specimen numbers, features, use Acceptance criteria Analytical sensitivity WHO First International Standard Human EBV DNA (09/260; 5 000 000 IU/vial) (or calibrated reference materials) NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimum 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device. LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit).³⁸ quantitative NAT: definition of lower, upper quantification limit, precision, accuracy, ‘linear’ measuring range, ‘dynamic range’. Reproducibility at different concentration levels According to the state of the art Diagnostic sensitivity EBV strain sensitivity Patient specimens determined as EBV DNA positive by comparator device Dilution series of EBV positive cell cultures may serve as potential substitutes Qualitative NAT: ≥100 Quantitative NAT: ≥100 dilution series for demonstration of quantification efficiencies Diagnostic specificity Negative specimens Qualitative NAT: ≥500 Quantitative NAT: ≥100 According to the state of the art Cross-reactivity Potentially cross-reacting specimens ≥20 specimens in total Including human specimens positive for related human herpesviruses, e.g. CMV, HHV6, VZV Herpesvirus positive cell cultures may serve as potential substitutes According to the state of the art Carry-over High EBV DNA positive; EBV DNA negative At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The virus titres of the high positive specimens shall be representative of high virus titres occurring naturally. According to the state of the art Whole system failure rate EBV DNA low-positive ≥100 EBV DNA low-positive specimens shall be tested. These specimens shall contain a virus concentration equivalent to three times the 95 % positive cut-off virus concentration. ≥99% positive

Including testing of other EBV markers and parameters (e.g. VCA-IgM, EBNA-1 IgG, immunoblot) or previous / follow-up specimens to assess the true specimen status.

Supplementary testing to confirm recent EBV infection: e.g. VCA-IgM, IgG-avidity, immunoblot analysis.

At an assumed EBV prevalence of 80 % corresponding to an initial number of 1000 donors.

Including pre-transplant recipients.

Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

ANNEX XI

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OF MARKERS OF TREPONEMA PALLIDUM INFECTION

Scope

This Annex applies to devices intended for detection of markers of Treponema pallidum (T. pallidum).

Table 1 applies to first-line assays for antibodies against T. pallidum (anti-T.pallidum).

Table 2 applies to confirmatory and supplemental anti-T.pallidum assays.

Table 1. First-line assays: anti-T.pallidum

Performance characteristic	Specimens	Specimen number, features, use	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥200 positive specimens in total, at different stages of the infection if available, including high positive and low positive specimens, identified as positive by at least two different serological tests (one of which is an enzyme immunoassay) for different antibodies to T.pallidum	≥99.5% overall sensitivity
Diagnostic sensitivity	Seroconversion panels	At least 1 seroconversion panel, ≥1 if possible, including individual specimens from the early infection phase	Diagnostic sensitivity during seroconversion shall represent the state of the art.
Analytical sensitivity	Standards	WHO international standards NIBSC code 05/132, when available
Diagnostic specificity	Unselected blood donors (including first-time donors)³⁹	≥5 000	≥99,5%
Diagnostic specificity	Hospitalised patients	≥200	Potential limitations for specificity, if any, shall be identified
Cross-reactivity	Potentially cross-reacting specimens	≥100 in total including the following specimens: positive for Borrelia burgdorferi sensu lato confirmed by IgG immunoblot; anti-HIV positive; RF+; other related microbial/infectious agents; systemic lupus erythematosus (SLE) patients; antiphospholipid antibody positive; pregnant women etc.	Potential limitations for specificity, if any, shall be identified

Table 2. Confirmatory and supplemental assays: anti-T.pallidum

Performance characteristic	Specimens	Specimen number, features, use	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥300 positive specimens at different stages of the infection (early primary syphilis, secondary stage, and during late syphilis ) including high positive specimens, 50 low positive specimens, by at least two different serological tests (one of which is an enzyme immunoassay) for different antibodies to T.pallidum	99% identification as “confirmed positive” or “indeterminate”
Diagnostic sensitivity	Seroconversion panels	At least 1 seroconversion panel, ≥1 if possible, including individual specimens from the early infection phase	Diagnostic sensitivity during seroconversion shall represent the state of the art
Analytical sensitivity	Standards	WHO international standards NIBSC code 05/132
Diagnostic specificity	Blood donors	≥200	≥ 99%;
Diagnostic specificity	Clinical specimens	≥200	Potential limitations for specificity, if any, shall be identified
Cross-reactivity	Potentially cross-reacting specimens	≥50 in total, including specimens from pregnant women and specimens with indeterminate results in other confirmatory assays.	Potential limitations for specificity, if any, shall be identified

Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donors.

ANNEX XII

COMMON SPECIFICATIONS FOR DEVICES INTENDED FOR DETECTION OR QUANTIFICATION OF MARKERS OF TRYPANOSOMA CRUZI INFECTION

Scope

This Annex applies for devices intended for detection or quantification of markers of Trypanosoma cruzi (T. cruzi) infection.

Table 1 applies to first-line assays for antibodies against T. cruzi (anti-T. cruzi).

Table 2 applies to confirmatory and supplemental anti-T. cruzi assays.

Table 3 applies to qualitative and quantitative NAT devices for T. cruzi DNA.

Table 1. First-line assays: anti-T. cruzi

Performance characteristic	Specimens	Specimen number, features, use	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥400 positive specimens, including highly positive confirmed by at least two different serological tests for different antibodies to T.cruzi. Of those 400, ≥25 parasite positive specimens which have been confirmed by direct detection.	99.5% overall sensitivity
Diagnostic sensitivity	Seroconversion panels	To be defined when available	Diagnostic sensitivity during seroconversion shall represent the state of the art
Analytical sensitivity	Standards	WHO international standards NIBSC code: 09/186 NIBSC code: 09/188
Diagnostic specificity	Unselected donors (including first-time donors)⁴⁰	≥5 000	≥99,5%
Diagnostic specificity	Hospitalised patients	≥200	Potential limitations for specificity, if any, shall be identified
Cross-reactivity	Potentially cross-reacting specimens	≥100 in total including the following specimens: positive for anti-Toxoplasma gondii; at least 5 specimens positive for anti-Leishmania; RF+; related microbial agents or other infectious agents; SLE patients; antiphospholipid antibody positive patients; pregnant women, etc.	Potential limitations for specificity, if any, shall be identified

Table 2. Confirmatory and supplemental assays: anti-T. cruzi

Performance characteristic	Specimens	Specimen number, features, use	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥300 positive specimens, including highly positive confirmed by at least two different serological tests for different antibodies to T.cruzi. Of those 300, ≥25 parasite positive specimens, which have been confirmed by direct detection.	≥99% identification as “confirmed positive” or “indeterminate”
Diagnostic sensitivity	Seroconversion panels	As available	Diagnostic sensitivity during seroconversion shall represent the state of the art, if applicable
Analytical sensitivity	Standards	WHO international standards NIBSC code: 09/186 NIBSC code: 09/188
Diagnostic specificity	Negative specimens	≥200	≥99%
Diagnostic specificity	Clinical specimens	≥200	Potential limitations for specificity, if any, shall be identified
Cross-reactivity	Potentially cross-reacting specimens	≥50 in total, including specimens from pregnant women and specimens with indeterminate results in other confirmatory assays	Potential limitations for specificity, if any, shall be identified

Table 3: NAT devices for T. cruzi DNA


	1.	For target sequence amplification devices, a functionality control for each specimen (internal control) shall reflect the state of the art. This control shall as far as possible be used throughout the whole process, i.e. extraction, amplification/hybridisation, detection.

---	---	---
	2.	Genotype and/or subtype detection shall be demonstrated by appropriate primer or probe design validation and shall also be validated by testing characterised genotyped specimens.

---	---	---
	3.	Potential cross-reactivity of non-target nucleic acid sequences shall be analysed by appropriate primer or probe design validation and shall also be validated by testing selected specimens.

---	---	---
	4.	Results of quantitative NAT devices shall be traceable to international standards or calibrated reference materials, if available, and be expressed in international units utilised in the specific field of application. Performance characteristic Specimens Specimen number, features, use Acceptance criteria Analytical sensitivity Characterized in-house reference preparation (as long as international reference materials are not available) NAT sensitivity and NAT LOD shall be validated by dilution series of reference materials, testing of replicates (minimal 24) at different analyte concentrations, including those with transition from positive to negative results with the respective NAT device. LOD shall be expressed as 95% positive cut-off value (IU/ml) after statistical analysis (e.g. Probit).⁴¹ According to the state of the art Diagnostic sensitivity: different T.cruzi strains / isolates Patient specimens from different regions determined as T.cruzi DNA positive by comparator device; sequence variants ≥100 Dilution series of T.cruzi positive cell cultures (isolates) or T.cruzi positive materials from animal models may serve as potential substitutes According to the state of the art Diagnostic specificity Negative specimens ≥100 According to the state of the art Cross-reactivity Potentially cross-reacting specimens ≥10 human specimens positive for other parasites, e.g. Plasmodium species, Trypanosoma brucei. Positive cell cultures may serve as potential substitutes According to the state of the art Carry-over At least five runs with alternating high-positive and negative specimens shall be performed during robustness studies. The T.cruzi titres of the high positive specimens shall be representative of high T.cruzi titres occurring naturally. According to the state of the art Whole system failure rate ≥100 T.cruzi DNA low-positive specimens shall be tested. These specimens shall contain a T.cruzi concentration equivalent to three times the 95 % positive cut-off T.cruzi concentration. ≥99% positive

Blood donor populations shall be investigated from at least two blood donation centres and consist of consecutive blood donations, which have not been selected to exclude first-time donors.

Reference: European Pharmacopeia 9.0, 2.6.21 Nucleic acid amplification techniques, Validation.

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

⁴² 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 seroconversion⁴³ (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 appropriate⁴⁴; consideration of genetic variants	≥90% sensitivity⁴⁵ for specimens taken >21 days after onset of symptoms⁴⁶; overall sensitivity including the early infection phase shall be at least equivalent to the comparator device⁴⁷
Diagnostic sensitivity	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 / quantitative⁴⁸ result output; RP: all antibody assays
Diagnostic specificity	Negative specimens⁴⁹	≥400 specimens from non-infected and non-vaccinated individuals⁵⁰	>99% specificity⁵¹
Diagnostic specificity		≥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.	Potential limitations for specificity, if any, shall beidentified

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	≥200⁵² Specimens⁵³ 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 freshly⁵⁴ vaccinated individuals if appropriate; consideration of genetic variants	≥80% sensitivity⁵⁵ for specimens taken during the first 21 days after symptom onset⁵⁶; overall sensitivity shall be at least equivalent to the comparator device⁵⁷ of the same type (i.e. IgM and/or IgA)
Diagnostic sensitivity	Seroconversion panels	As far as available	Seroconversion sensitivity comparable to other CE-marked devices
Analytical sensitivity	Standards	N/A	N/A
Diagnostic specificity	Negative specimens⁵⁸	≥200 specimens from non-infected and non-vaccinated individuals⁵⁹	≥98% specificity⁶⁰
Diagnostic specificity		≥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.	Potential limitations for specificity, if any, shall be identified

Table 3: Confirmatory or supplemental

⁶¹ 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”)
Diagnostic sensitivity	Seroconversion panels/low titre panels	as far as available	Correct determination as “positive” (or “indeterminate”)
Analytical sensitivity	Standards	N/A	N/A
Diagnostic specificity	Negative specimens⁶²	≥200 from non-infected / non-vaccinated population	No false positive results; correct determination as “negative” (or “indeterminate”)
Diagnostic specificity		≥200 from hospitalised patients (without SARS-CoV-2 infection)	No false positive results; correct determination as “negative” (or “indeterminate”)
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	No false positive results; correct determination as “negative” (or “indeterminate”)

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

Performance characteristic	Specimen	Specimen number, features, use	Acceptance criteria
Diagnostic sensitivity	Positive specimens	≥100⁶³ NAT positive specimens⁶⁴ from early infection within the first 7 days after symptom onset⁶⁵; specimens shall represent naturally occurring viral loads⁶⁶; consideration of genetic variants⁶⁷; consideration of variations in specimen collection and/or specimen handling⁶⁸	Detection of >80% (rapid tests); detection of >85% (lab-based assays⁶⁹); relative to SARS-CoV-2-NAT⁷⁰,⁷¹
Analytical sensitivity	Standards	As soon as available	Establishment of a LOD⁷²
Diagnostic specificity	Negative specimens	≥300 from non-infected individuals	Specificity >98% (rapid tests) Specificity >99% (lab-based assays⁶⁹)
Diagnostic specificity	Negative specimens	≥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 bacteria⁷³ present in the specimen taking area	Potential limitations for specificity, if any, shall be identified

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

Performance characteristic	Specimen	SARS-CoV-2 RNA qualitative	SARS-CoV-2 RNA quantitative
Sensitivity	Sensitivity	Sensitivity	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	≥100⁷⁴
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 analysis⁷⁵; 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	Specificity	Specificity	Specificity
Diagnostic specificity	SARS-CoV-2 RNA negative human specimens	≥500	≥100
In silico analysis⁷⁵		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	Robustness	Robustness	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

⁷⁶

Performance characteristic	Specimens⁷⁷	Number of lay persons
Result interpretation⁷⁸	Interpretation of results⁷⁹ by lay persons reflecting the following range of reactivity levels: — non-reactive — reactive — weak reactive⁸⁰ — invalid	≥100
Diagnostic sensitivity⁸¹	Lay persons that are known antigen positive⁸² ^, ⁸³	≥30
Diagnostic specificity⁸⁴	Lay persons that do not know their status⁸⁰	≥60

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

⁸⁵

Performance characteristic	Specimens⁸⁶	Number of lay persons
Result interpretation⁸⁷	Interpretation of results⁸⁸ by lay persons reflecting the following range of reactivity levels: — non-reactive — reactive — weak reactive⁸⁹ — invalid	≥100
Diagnostic sensitivity⁹⁰	Lay persons that are known antibody positive⁹¹	≥100
Diagnostic specificity⁹²	Lay persons that do not know their status⁸⁹	≥100

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

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

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

Based on confirmed positive SARS-CoV-2 NAT result.

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.

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

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

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

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

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

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

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

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

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.

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

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

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

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).

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

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

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.

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.

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

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.

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.

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.

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

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.

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.

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.

E.g. staphylococci and streptococci expressing protein A or G.

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

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.

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

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.

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.

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

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

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.

Subjects up to about 7 days after symptom onset.

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

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

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

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.

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

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