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Annals of Pediatrics and Child Health

Performance of Multiplex Detection Method of IgM Class Antibodies against Toxoplasma gondii, Rubella and Human Cytomegalovirus

Research Article | Open Access Volume 10 | Issue 7 |

  • 1. Instituto de Pesquisa e Desenvolvimento (IP&D), Universidade do Vale do Paraíba (UNIVAP), Brasil.
  • 2. Intercientífica, Parque Tecnológico UNIVAP, Brasil
  • 3. Instituto de Ciência e Tecnologia, Universidade Estadual Paulista (UNESP), Brasil
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Corresponding Authors
Rainara Moreno Sanches de Almeida, Instituto de Pesquisa e Desenvolvimento (IP&D), Universidade do Vale do Paraíba, Av. Shishima Hifumi 2911, São José dos Campos, São Paulo, Brasil, Tel: 55(12)988036744
ABSTRACT

Serological diagnosis during neonatal screening is crucial in disease prevention. Among the infectious diseases, the most common are toxoplasmosis, rubella, and cytomegalovirus. Traditional diagnostic methods are used to detect a single infectious agent per test. The use of multiplex detection methods increases productivity and reduces the amount of material used, resulting in a more efficient test from a technical, environmental, and economic point of view. The study’s objective was to evaluate the performance of a new diagnostic method aimed at neonatal screening using the multiplex platform of magnetic microspheres from the company Luminex Corporation. For this, tests were carried out for analytical validation of the diagnostic product developed following the rules of the National Health Surveillance Agency (ANVISA) of Brazil. The parameters evaluated were repeatability, reproducibility, linearity, robustness, high dose, minimum detection limit, and analytical specificity. All data obtained met the acceptance criteria of RDC 166/17 of 2017 for the use of the diagnostic product in the national territory. Repeatability and reproducibility tests showed a CV of less than 15% between replicates of the same operator and different operators. The kit showed linearity throughout the operating range with R2 above 0.990, and no effect of high-efficiency dose was observed in the chosen working dilution. In addition, the kit did not show interference from the matrix with the results, and it was observed that small and deliberate changes in the incubation time of each reagent did not have a significant effect on the data obtained.

KEYWORDS

Neonatal screening, Multiplex assay, Magnetic beads, Analytical validation

CITATION

de Almeida RMS, Ogawa GM, Siqueira Custódio PR, Socorro Sampaio MA, da Silva NS, et al. Performance of Multiplex Detection Method of IgM Class Antibodies against Toxoplasma gondii, Rubella and Human Cytomegalovirus. Ann Pediatr Child Health 2022; 10(7): 1295.

ABBREVIATIONS

ANVISA: National Health Surveillance Agency; RDC: Resolution of the collegiate board; CV: Variation Constance; TOX: Toxoplasmosis; RUB: Rubella; CMV: Cytomegalovirus.

INTRODUCTION

The detection of antibodies against infectious diseases is an activity widely used in several situations. Among the available laboratory detection techniques, the widely used method is the Enzyme-Linked Immunosorbent Assay (ELISA) which detects a single infectious agent by an assay based on antigen-antibody binding [1].

Although commonly used, traditional methods of serological diagnoses, such as ELISA, have the disadvantage of requiring more significant amounts of samples and time for analysis since each analyte of interest requires a specific assay.

New technologies were developed to optimize diagnostic methods, among which stands out is the xMAP® technology from Luminex Corporation, which allows the simultaneous analysis of several parameters in the same biological sample using microspheres [2]. Neonatal screening is necessary to prevent severe infections from affecting the fetus and neonate.

Several infectious agents could be vertically transmitted from mother to fetus via the transplacental route or direct contact during childbirth. TORCH’s acronym represents the most common infections, including toxoplasmosis, rubella, and cytomegalovirus [3,4].

A multiplex kit was developed for detecting antibodies against infectious diseases using xMAP® technology of magnetic microspheres, aiming to meet the need to develop effective diagnostic methods for neonatal screening. The kit is intended to detect multiple and simultaneous anti-toxoplasmosis, anti-rubella, and anti-human cytomegalovirus IgM antibodies in newborn samples.

Developing a new diagnostic product using microspheres demands studies on the performance and other parameters that an in vitro diagnostic kit must present to be used in Brazil. The National Health Surveillance Agency (ANVISA) created and evaluated these performance parameters.

For the validation and registration of the in vitro diagnostic kit and its entry into the national market, different studies are required following the rules of the Resolution of the Collegiate Board (RDC) No. 166 of July 24, 2017, published by ANVISA, which establishes the criteria for validation of analytical methods.

The objective of the present study was to evaluate the performance following the analytical validation criteria of a new diagnostic method for multiple and simultaneous detections of human antibodies of the IgM class against Toxoplasma gondii (TOX), rubella (RUB) and cytomegalovirus (CMV) in the field of newborn screening.

MATERIALS AND METHODS

Performance analysis

For the use of new diagnostic methods developed in Brazil, the product must meet ANVISA’s analytical methods validation criteria. RDC No. 166 of July 24, 2017, applies to analytical methods used in pharmaceutical supplies, medicines, and biological products in all their production stages. Some validation parameters involve precision (repeatability and reproducibility), Analytical Specificity, High Dose, Linearity, Limit of Detection (LOD), and Robustness [5]. All tests carried out in the present study were based on the acceptance criteria for registering a new diagnostic product in Brazil and its use in the national territory.

Characterization and validation of clinical samples used.

Reference samples purchased from Seracare Life Science were used for the three parameters. These samples are Seracare’s antibodies panel: antibody anti-T. gondii antibody panel (AccuSet™ Toxoplasmosis Performance Panel, catalog number 0820- 0321, lot 10344475), antibody IgM carrier sample Rubella (Seracare Rubella IgM positive plasma, catalog number DS-674 -M, lot 9254456), and antibodies carrying IgM anti-CMV (Seracare CMV IgM Positive plasma, catalog number DS-626-M, lot BM217341), all samples were supplied with a certificate of analysis and reference results from other methodologies.

Coupling of antigens to magnetic microspheres

Couplings were performed following the protocol provided by Luminex Corporation. (xMAP® Cookbook 5th Edition). First, the xMAP® magnetic microsphere solutions were added in different low-adhesion USA tubes (catalog 1415-2600). Microspheres 12 (MC10012), 45 (MC10045), and 72 (MC10072) were used for Toxoplasma gondii, Rubella, and Cytomegalovirus antigens, respectively. The microspheres were washed with NaH2PO4 (activation buffer) and incubated with EDC and Sulfo NHS for 20 minutes, protected from light, and on rotation in a tube shaker at 700 rpm. Then the microspheres were washed with PBS-TBN and incubated with the respective antigens for 2h. After incubation, the microspheres were washed and kept in rotation for 20 minutes with block buffer (PBS/1%BSA). Then the microspheres were centrifuged, resuspended in PBS-TBN buffer, and stored under refrigeration.

For reasons of commercial secrecy, the antigens and concentrations used cannot be made available. All couplings make up reagent 2 of the NeoMAP® 3plex IgM kit from Intercientífica, developed in this study, with registration number 80173700020 at ANVISA.

Assay protocol

First, the samples were eluted in 1.5 ml tubes with 200 µL of Elution Reagent (PBS, Tween20, 20% Azide, BSA, and E. coli extract) at a dilution of 1:200 for 60 minutes under agitation at 700rpm. After incubation, the assay plate was prepared by adding 25 µL of the pool of magnetic microspheres coupled to Toxoplasmosis, Rubella, and Cytomegalovirus antigens in each well. Then a washing step was performed by adding 50 µL of Washing Solution (PBS, Tween20, and 20% Azide). Next, 50 µL of the eluate was added to the wells and incubated for 90 minutes under agitation at 700 rpm, protected from light. After incubation, two washing steps were performed, and 50 µL of Anti-human IgM solution labeled with Biotin was added for 30 minutes under agitation on the plate shaker and protected from light. After the incubation, a new washing step was performed, and 50 µL of the Phycoerythrin solution conjugated with Streptavidin was added and maintained under agitation for 30 minutes in the plate shaker at 700 rpm. Then the solution was removed, 50 µL of Washing Solution was added and kept for 1 minute in the plate shaker, taken for reading in the Magpix equipment with analysis in the xPONENT® Software. For reasons of commercial secrecy, the antigens and concentrations used cannot be made available. As well as the Anti-Human IgM and Phycoerythrin conjugated with Streptavidin catalogs. All reagents make up the NeoMAP® 3plex IgM kit developed in this study and with registration 80173700020 at ANVISA.

Analysis of results

In all tests, the constant of variation (CV) between replicates of each sample was verified. For this, the formula below was used (Figure 1). In addition, GraphPad Prisma and Action Stat software were used for the statistical analysis of the data.

Figure 1 Formula for constant of variation (CV).

Figure 1: Formula for constant of variation (CV).

Measurement accuracy

The repeatability tests were carried out in the Research and Development laboratory of the company Intercientifica, and the reproducibility tests were carried out in the Quality Control laboratory in another sector of the same company, where all the equipment and the operator were different. The assays were carried out with nine high, medium, and low reactivity samples in triplicate and on other days.

Minimum Limit of Detection (LOD)

The LOD Limit of Detection must be demonstrated by obtaining the lowest amount of the analyte present in a sample that can be detected but not necessarily quantified under established experimental conditions. For this, 73 measurements of the blank were performed, with the blank consisting of all reagents, except for the sample. Statistical analysis was performed using the Action Stat software, which presented the minimum detection values for each parameter in the multiplex assay.

Analytical Specificity

Specificity was assessed by the method’s ability to unambiguously identify or quantify the analyte of interest in the presence of components that may be present in the sample, such as matrix components. The test protocol was carried out using the blood components after separating the plasma in triplicate to verify if matrix interference occurs because it is in the plasma that the antibodies are found. Data analysis was performed using the formula below (figure 2), which considers the results in Mean Fluorescence Intensity (MFI) and the blank.

Figure 2 Formula used for analytical sensitivity analysis.

Figure 2: Formula used for analytical sensitivity analysis.

Linearity

The linearity of the method was verified through tests with highly positive samples for each parameter in serial dilution 1:2 with 16 drops. After the trial, linear regression analysis was performed on each sample, evaluating the regression coefficient (R2). The defined performance target was R2 greater than 0.99 according to ANVISA’s acceptance criteria.

High Dose

The high dose prozone effect was verified using highly reactive samples for the three antigens. The test was carried out with samples in serial dilution 1:2 with eight drops starting with the dilution of the samples in 1:10.

Robustness

To verify whether small, deliberate changes in some steps cause changes in the reactivity of the samples. Tests were carried out with 10 minutes more and 10 minutes less than the standard incubation time of each step of the test protocol: sample elution, incubation with the microspheres, incubation with Antihuman IgM, and incubation with phycoerythrin. The acceptance criterion was a CV of less than 20% between samples with different incubation times compared to the reference (original time of each stage).

RESULTS

Measurement Accuracy

The repeatability test (Table 1), showed a CV of less than 15% in all samples, demonstrating no significant difference between triplicates for all analyzed parameters.

Table 1: Repeatability analysis.

TOX

Sample

Well 1

Well 2

Well 3

M

SD

CV%

1

25737

27455

27450

27450

809

3

2

22508

25824

26491

25824

1742

7

3

8076

8626

8785

8626

304

3

4

3331

3929

4326

3929

409

10

5

2737

3514

3644

3513

400

11

6

3476

2967

3163

3163

210

7

7

370

376

319

370

26

7

8

346

336

324

336

9

3

9

287

203

254

254

34

13

RUB

Sample

Well 1

Well 2

Well 3

M

SD

CV%

1

28094

30167

30621

30167

1100

4

2

26494

26115

25568

26115

380

1

3

20084

22046

21360

21360

813

5

4

15956

14871

15745

15745

470

3

5

10074

10350

9518

10074

346

3

6

6011

5815

5762

5815

107

2

7

3171

3113

2856

3113

137

4

8

1584

1542

1122

1542

209

13

9

773

732

668

732

43

6

CMV

Sample

Well 1

Well 2

Well 3

M

SD

CV%

1

14934

15109

14604

14934

210

1

2

12386

12066

12469

12386

174

1

3

11111

12947

12580

12580

793

6

4

7422

7575

7471

7471

64

1

5

7546

8394

8094

8094

351

4

6

4402

4812

4066

4402

305

7

7

1236

1156

1151

1156

39

3

8

896

892

869

891

11

1

9

579

569

580

579

5

1

Abbreviations: M: medium; SD: Standard deviation; CV: Constance of variation.

Although the acceptance criterion used was 15%, it is common for low reactivity samples to present a relative standard deviation above the recommended one due to the intrinsic variability of the method resulting from the high dynamic range (0-100,000MFI).

In Table 2, it is possible to observe CV less than 15% in all parameters when comparing the tests performed by two operators in two different laboratories.

Table 2: Reproducibility Analysis.

TOX

Sample

Well 1

Well 2

Well 3

M lab 2

SD

CV (%)

M lab 1

M Lab 1 vs. Lab 2

SD

CV (%)

1

22495

23908

22571

22571

649

3

27450

25010

2440

10

2

22222

23608

24019

23608

769

3

25824

24716

1108

4

3

8021

7750

7951

7951

115

1

8626

8288

338

4

4

4158

4277

4107

4158

71

2

3929

4043

114

3

5

2761

2775

2446

2761

152

5

3514

3137

377

12

6

2616

2536

2326

2536

122

5

3163

2849

314

11

7

639

646

672

647

14

2

724

685

39

6

8

323

302

325

323

11

3

370

347

24

7

9

352

380

308

352

30

8

336

344

8

2

RUB

Sample

Well 1

Well 2

Well 3

M lab 2

SD

CV (%)

M lab 1

M Lab 1 vs. Lab 2

SD

CV (%)

1

33277

36123

32433

33277

1579

5

30167

31722

1555

5

2

28718

30542

29543

29543

746

3

26115

27829

1714

6

3

24138

24113

23756

24113

174

1

21360

22736

1376

6

4

18332

16409

18236

18236

885

5

15745

16990

1245

7

5

12423

12187

12073

12187

146

1

10074

11130

1056

9

6

7573

6998

7270

7270

235

3

5815

6543

727

11

7

3844

3891

3921

3891

32

1

3113

3502

389

11

8

1868

1852

1545

1852

148

8

1542

1697

155

9

9

934

868

828

868

44

5

732

800

68

8

 

CMV

Sample

Well 1

Well 2

Well 3

M lab 2

SD

CV (%)

M lab 1

M Lab 1 vs. Lab 2

SD

CV (%)

1

15154

14815

14775

14815

170

1

12386

13601

1214

9

2

13316

16048

16051

16048

1289

8

12580

14314

1734

12

3

12101

12565

12182

12182

203

2

11259

11720

462

4

4

6373

6591

6306

6373

122

2

7471

6922

549

8

5

9202

8711

8484

8711

300

3

8094

8402

308

4

6

5041

4784

4372

4784

275

6

4402

4593

191

4

7

3385

3243

3306

3306

58

2

2771

3038

268

9

8

1188

1071

1115

1115

48

4

1156

1135

20

2

9

510

526

479

510

19

4

579

544

34

6

Abbreviations: Results from labs 1 and 2 with median, standard deviation, and CV of nine samples analyzed with the NeoMAP® 3plex IgM kit (M- Median, SD- standard CV – Constant Variation, Op- operator, Lab- laboratory).

Limit of Detection (LOD)

In all parameters, there was low reactivity in the blank, indicating no undesired reactivity of the elution and washing buffer with the microspheres coupled to the different antigens (Table 3).

Table 3: Limit of Detection in MFI of the NeoMAP® 3plex IgM Kit.

Limit of Detection (LOD)

 

TOX

RUB

CMV

Average

36

49

41

Standard deviation

5

9

4

Degrees of liberty

71

71

71

Detection limit

44

65

47

Analytical specificity

Table 4 demonstrates that the reactivity of blood components in the absence of antibodies is below the detection limits of all parameters, indicating no significant reactivity of these components.

Table 4: Analytical specificity.

 

TOX

RUB

CMV

Replica 1

2

0.5

0

Replica 2

0

-4.5

-5

Replica 3

1

1.5

-4

High Dose

In the figure below (Figure 3), it is possible to see that in the TOX parameter, a high dose effect occurs from the dilution of 1/40, causing a decrease in reactivity even with a higher concentration of antibodies. The RUB parameter occurs from the 1/160 dilution, and the CMV parameter from the 1/20 dilution.

Figure 3 Reactivity curve of the samples in relation to the dilution factor in the parameters of TOX, RUB, and CMV. (A) TOX, (B) RUB and (C) CMV.

Figure 3: Reactivity curve of the samples in relation to the dilution factor in the parameters of TOX, RUB, and CMV. (A) TOX, (B) RUB and (C) CMV.

Linearity

The response range is linear and occurs between 15 - 10,000 MFI with R2 and 0.99 correlation for TOX, 170 - 3,000 MFI with R2, 0.99 correlation for RUB, and 10 - 6,000 MFI with R2 and 0 correlation, 99 for CMV. At MFI values above or below these ranges, the test cannot correlate MFI with the concentration unit of the samples.

Table 5: Reactivity of samples incubated 80, 90 and 100 minutes in the elution step.

TOX

80min

90min

100min

M.

SD.

CV (%)

1

615

623

615

615

4

1

2

21279

22669

21397

21396

629

3

3

1610

1629

2080

1629

217

13

4

507

518

572

518

28

5

RUB

80min

90min

100min

M.

SD.

CV (%)

1

27770

25990

26267

26267

782

3

2

181

249

222

222

28

13

3

329.5

389

423

389

38

10

4

53

65

60

60

5

8

CMV

80min

90min

100min

M.

SD.

CV (%)

1

453

552

457

457

46

10

2

3033

4540

3222

3222

670

21

3

9208

9778

12205

9778

1299

13

4

300

411

382

382

47

12

Abbreviations: M: Medium; SD: Standard deviation; CV: Constant of variation.

Robustness

Robustness studies indicate that variations of ten minutes plus or minus in incubations do not result in significant differences in the reactivity of samples with an overall CV of less than 15%. The results are in the tables below (Tables 5, 6, and 7).

Table 6: Reactivity of samples incubated 20, 30 and 40 m minutes in the secondary antibody step.

TOX

20min

30min

40min

M.

SD.

CV (%)

1

640

623

698

640

32

5

2

18606

22669

22793

22669

1945

9

3

1717

1629

1907

1717

116

7

4

536

518

599

536

35

6

RUB

20min

30min

40min

M.

SD.

CV (%)

1

25301

25990

27931

25989

1113

4

2

188

249

230

230

25

11

3

393

389

423

393

15

4

4

60

68

68

65

3

5

CMV

20min

30min

40min

M.

SD.

CV (%)

1

493

552

508

507

25

5

2

2840

4540

4099

4098

720

18

3

10352

9778

10966

10351

485

5

4

386

411

419

411

14

3

Abbreviations: M: Medium; SD: Standard deviation; CV: Constant of variation.

Table 7: Reactivity of samples with incubation of 20, 30 and 40 minutes in the phycoerythrin step

TOX

20min

30min

40min

M.

SD.

CV (%)

1

727

623

566

623

67

11

2

19032

22669

22974

22669

1791

8

3

1803

1629

1816

1803

85

5

4

567

518

623

567

43

8

RUB

20min

30min

40min

M.

SD.

CV (%)

1

25175

25990

28076

25990

1222

5

2

203

249

221

221

19

9

3

362

389

376

376

11

3

4

68

65

73

68

3

5

CMV

20min

30min

40min

M.

SD.

CV (%)

1

519

552

456

518

40

8

2

3392

4540

4200

4200

482

12

3

9879

9778

12273

9879

1153

12

4

379

411

415

411

16

4

Abbreviations: M: Medium; SD: Standard deviation; CV: Constant of variation.

DISCUSSION

Since the introduction of neonatal screening, several technological advances have been used for the early detection of congenital conditions. Implementing new methodologies allows for a significant increase in the number of diseases screened, with the capacity to include up to fifty disorders [5].

Recent technological advances have developed platforms capable of multiplexing multiple molecular and immunological assays for high-throughput screening. It is a modern platform whose main advantages are saving time and reagents [5].

Among the new technologies, the multiplex platform of magnetic microspheres from the company Luminex Corporation allows the development of assay methods with up to 100 different analytes in the same well of the assay plate [6].

Analytes must be coupled to these microspheres to perform target detection. The coupling is carried out by the chemical interaction of the carboxyl groups on the microspheres’ surfaces with the proteins’ primary amines. These bonds are covalent and most often performed with antibodies or specific antigens [8,9].

In general, these microspheres are used in scientific research, and there needs to be more information about them in neonatal screening. Screening has the potential to prevent serious health problems, including death. Worldwide, neonatal screening programs have evolved from simple tests to comprehensive and complex systems capable of detecting more than 50 different conditions in different countries [9].

The screening program is used to track diseases and mainly to seek treatment and provide the family with resources so that those affected become healthy. In Brazil, more and more diseases are being implemented in the Unified Health System (SUS) screening programs, including the addition of infectious diseases such as Toxoplasmosis, Rubella, and Cytomegalovirus correspond to a high number of severe congenital infections [10].

With the expansion of screened diseases, developing new technologies that optimize and implement analyses in all regions, including the country’s most remote regions, is essential. For the use of these new diagnostic methods developed in Brazil, the product must meet ANVISA’s acceptance criteria for validation of analytical methods.

The precision of an analytical procedure expresses the proximity of agreement (degree of dispersion) between a series of measurements obtained from the same sample [11]. In this study, the repeatability and reproducibility assays performed for precision analysis showed a CV of less than 15% in all analyzes, demonstrating agreement between different tests with the same sample. The kit under development is considered semiquantitative. This definition occurs because the kit qualitatively delivers the results, that is, positive or negative. However, occasionally quantitative values can be used, mainly in the case of recollection of the same patient.

The MFI values of a result can be compared with results from a later collection (from the same individual) to verify whether there has been a significant increase or decrease in reactivity.

Thus, it is necessary to demonstrate that the MFI response of the product is proportional to the concentration of antibodies in the samples.

The linearity of the method is the ability to obtain results directly proportional to the concentration of the specific target antibody against the analyte of interest in the sample [12].

Our study verified linearity using samples with data on reactivity units. As these samples have unit values, it is possible to verify how the response occurs about the dose: MFI and AU/ mL or s/co ratio. The tests indicate that all tested parameters can produce a response (in MFI) corresponding to the dosage of IgM antibodies in the reference samples.

The high dose prozone effect was verified using highly reactive samples for the three antigens. In the assay, it was possible to observe an effect of a high dose at the highest concentrations with a decrease in reactivity. However, this decrease did not occur significantly with CV less than 15% when comparing the MFI values. Furthermore, although there is inhibition of antigenantibody binding, it is far from the dilution used in the study.

All parameters showed low LOD values, with 44, 65, and 47 MFI, respectively, for TOXO, RUB, and CMV. LOD corresponds to the lowest concentration of the analyte that can be detected, but not necessarily quantified, under established experimental conditions; this is because, usually, an assay cannot measure analyte concentrations down to zero [12].

Analytical specificity is the ability to unambiguously evaluate the analyte in the presence of other components that may be present in the samples, such as the matrix. The method under development must be able to produce a response only for the specific target [13].

In the study, the NeoMAP® 3plex IgM kit proved specific in all parameters, with no matrix reactivity in the assay performed only with the red blood cell concentrate. That is, the kit could generate interference-free signals on the multiplex platform. Validation is fundamental for the efficient operation of new analytical methods and must be carried out at all stages, from the raw materials used to the finished product. The phrase validation implies a feasibility demonstration activity and aims to demonstrate that the developed product meets the recommended acceptance criteria and is suitable for the proposed objectives [10,11]. In the present study, the kit developed followed all the criteria proposed for product registration in Brazil and its use in the national territory.

CONCLUSION

The repeatability and reproducibility tests showed a CV of less than 15% in all samples, demonstrating the reliability of the data obtained in all tests. High-dose assays showed that at higher concentrations, there is a tendency to inhibit antigen-antibody binding, requiring a defined dilution of 1:200. The obtained data presented performance compatible with the validation criteria of analytical methods of ANVISA, being approved for use in the country. The data generated in this study are contained in the product registration (Registration number 80173700020).

ACKNOWLEDGMENTS

Acknowledgments to the Coordination for the Improvement of Higher Education Personnel (CAPES) for the master’s scholarship that enabled the development of the study. Acknowledgments to the company Intercientifica for sharing the infrastructure of the Research and Development laboratory and for making the xMAP technology of magnetic microspheres available.

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Received : 24 Nov 2022
Accepted : 19 Dec 2022
Published : 23 Dec 2022
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