95 Inviting comments on standard IVD evaluation protocols drafted by ICMR and CDSCO 2025-Jan-02 2843 KB

ICMR-CDSCO/IVD/GD/PROTOCOLS/01/2024

Indian Council of Medical Research and Central Drugs Standard Control Organization
Department of Health Research and Directorate General of Health Services
Ministry of Health and Family Welfare Government of India Document No.: ICMR-CDSCO/IVD/GD/PROTOCOLS/01/2024

Subject: Inviting comments on standard IVD evaluation protocols drafted by ICMR and CDSCO

Licensure of In-Vitro Diagnostics (IVDs) under Medical Devices Rules 2017 requires a detailed evaluation protocol for the performance evaluation of IVDs to evaluate their quality and performance. To facilitate this process, the Indian Council of Medical Research (ICMR) and CDSCO have come together to draft standard evaluation protocols for use by IVD manufacturers testing labs in India. Currently, the following IVD evaluation protocols have been developed by ICMR and CDSCO:

1. Performance evaluation protocol for Chikungunya IgM ELISA
2. Performance evaluation protocol for Chikungunya IgM RDT
3. Performance evaluation protocol for Chikungunya real-time PCR
4. Performance evaluation protocol for Dengue NS1 RDT
5. Field evaluation protocol for Dengue NS1 RDT
6. Performance evaluation protocol for Dengue NS1 ELISA
7. Field evaluation protocol for Dengue NS1 ELISA
8. Performance evaluation protocol for Dengue IgM RDT
9. Performance evaluation protocol for Dengue IgM ELISA
10. Performance evaluation protocol for Dengue NS1/ IgM combo RDT
11. Field evaluation protocol for Dengue NS1/ IgM combo RDT
12. Performance evaluation protocol for Dengue real-time PCR
13. Field evaluation protocol for Dengue real-time PCR
14. Performance evaluation protocol for Zika virus real-time PCR

The protocols are now being placed in the public domain for comments from relevant stakeholders.

rotocol for Dengue real-time PCR 14. Performance evaluation protocol for Zika virus real-time PCR

The protocols are now being placed in the public domain for comments from relevant stakeholders. This window of opportunity will close on 15th February 2025, and, once finalized, there will be minimal scope for change in these documents. Therefore, all interested stakeholders are requested to provide their comments before 15th February 2025, at ivdevaluation@gmail.com as per the enclosed format. Once the public consultation period concludes, all comments will be reviewed and considered in finalizing the draft protocols before final clearance by ICMR and CDSCO.

ICMR-CDSCO/IVD/GD/PROTOCOLS/01/2024

Dated: 31st December 2024 Place: New Delhi

STANDARD IVD PERFORMANCE EVALUATION PROTOCOL STAKEHOLDER FEEDBACK FORM

S.N. Name of the Protocol Document No. Page No. Line No. Current Text Proposed Text Explanation/Reference

Name:

Designation and Affiliation:

1 ARBOVIRUS IN-VITRO DIAGNOSTICS 2 ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 3 -Dengue virus, Chikungunya virus, Zika virus 4

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6 STANDARD PERFORMANCE EVALUATION PROTOCOLS
DRAFT FOR STAKEHOLDER COMMENTS DECEMBER, 2024 New Delhi, India

VD/GD/PROTOCOLS/02/2024 3 -Dengue virus, Chikungunya virus, Zika virus 4

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6 STANDARD PERFORMANCE EVALUATION PROTOCOLS
DRAFT FOR STAKEHOLDER COMMENTS DECEMBER, 2024 New Delhi, India

Arbovirus IVD Performance Evaluation Protocols ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 1 | Page

7 Table of Contents 8 S.No. Content Page Number 1.
Chikungunya IgM ELISA – Performance evaluation protocol 2 2.
Chikungunya IgM RDT – Performance evaluation protocol 10 3.
Chikungunya real time PCR – Performance evaluation protocol 18 4.
Dengue NS1 RDT – Performance evaluation protocol
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Dengue NS1 RDT – Field evaluation protocol
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Dengue NS1 ELISA – Performance evaluation protocol 43 7.
Dengue NS1 ELISA – Field evaluation protocol 51 8.
Dengue IgM RDT – Performance evaluation protocol 59 9.
Dengue IgM ELISA – Performance evaluation protocol 68 10.
Dengue NS1/IgM combo RDT – Performance evaluation protocol 76 11.
Dengue NS1/IgM combo RDT – Field evaluation protocol 86 12.
Dengue real time PCR – Performance evaluation protocol 95 13.
Dengue real time PCR – Field evaluation protocol 104 14.
Zika virus real time PCR – Performance evaluation protocol 112 15.
Information on operational and test performance characteristics required from manufacturers 121

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Performance evaluation protocol for Chikungunya IgM ELISA kits 20 I. Background:
21 CDSCO and ICMR, New Delhi, have aimed at facilitating the availability of Quality-Assured 22 diagnostic kits appropriate for use in India. Hence the following guidelines shall establish 23 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 24 evaluation is to independently verify the manufacturer’s claim regarding IVD performance. 25 II. Purpose: 26 To evaluate the performance characteristics of Chikungunya IgM ELISA kits in the diagnosis of 27 Chikungunya infection. 28 III. Requirements:
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1. Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 30 the kit to be evaluated works in a closed system format, the manufacturer needs to supply 31 the required equipment. 32
2. Evaluation sites/laboratories (With required equipment) 33
3. Reference test kits 34
4. Characterised Evaluation panel 35
5. Laboratory supplies
   36 IV. Ethical approvals:
   37 Exempted from Ethics approval as per ICMR’s Guidance on Ethical Requirements for Laboratory 38 Validation Testing, 2024. A self-declaration form as provided in ICMR guidelines to be submitted 39 by the investigators to the institutional authorities and ethics committee for information.

38 Validation Testing, 2024. A self-declaration form as provided in ICMR guidelines to be submitted 39 by the investigators to the institutional authorities and ethics committee for information. 40 V. Procedure: 41

1. Study design/type: Diagnostic accuracy study using archived/leftover clinical samples. 42
2. Preparation of Evaluation sites/laboratories:
   43 Identified IVD kit evaluation laboratories should establish their proficiency through
   44 A. Accreditation from NABL for at least one of the Quality management system (NABL 45 accreditation for testing Lab / calibration lab (ISO/IES 17025), Medical Lab (ISO 15189), PT 46 provider ISO/IEC 17043 or CDSCO approved Reference laboratory. 47 B. Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 48 and competency testing on following
   49 ⮚ Preparation & characterization of kit evaluation panel
   50 ⮚ Handling of Chikungunya IgM ELISA kits received for performance evaluation 51 (Verification/Storage/Unpacking etc). 52

wing
49 ⮚ Preparation & characterization of kit evaluation panel
50 ⮚ Handling of Chikungunya IgM ELISA kits received for performance evaluation 51 (Verification/Storage/Unpacking etc). 52

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⮚ Testing, interpreting, recording of results & reporting 53 ⮚ Data handling, data safety & confidentiality 54 3. Preparation of Chikungunya IgM ELISA IVD kit evaluation panel 55 Well characterised Chikungunya IVD kit evaluation panel is a critical requirement for performance 56 evaluation of IVD kits. Hence statistically significant number of sera samples should be available 57 from Chikungunya confirmed cases. Further characterised for Chikungunya IgM positivity by 58 using approved reference kits having high sensitivity and specificity. 59 Chikungunya IgM performance evaluation panel need to be tested again by the reference assays at 60 the time of evaluating a particular index test to confirm the positive and negative status of the 61 samples. 62 4. Reference assay:
63 All the samples will be tested by CDC/NIV real-time (RT-PCR) assay. Samples which are positive 64 by RT-PCR assay will be further tested by any two of the following IgM ELISA kits: 65 i. ICMR-NIV MAC ELISA kit 66 ii. Inbios CHIKjj Detect™ IgM ELISA 67 iii. Anti-Chikungunya virus ELISA (IgM) Test (Euroimmun, Luebeck, Germany) 68 Samples positive by at least two kits will be considered.

MAC ELISA kit 66 ii. Inbios CHIKjj Detect™ IgM ELISA 67 iii. Anti-Chikungunya virus ELISA (IgM) Test (Euroimmun, Luebeck, Germany) 68 Samples positive by at least two kits will be considered. If sufficient RT-PCR positive samples 69 are not available, samples positive by at least 2 ELISA kits (of the kits mentioned above) can 70 be considered as true positive samples.
71 Samples which are negative by RT-PCR and at least two IgM ELISA kits mentioned above will be 72 considered as Chikungunya negative samples. 73 5. Sample size and sample panel composition: Sample sizes of positive and negative 74 samples and sample panel composition against different values of sensitivity and specificity are 75 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of 76 significance, and an absolute precision of 5%. Appropriate sample size has to be chosen from the 77 tables according to the values of sensitivity and specificity being claimed by the manufacturer. If 78 a claimed sensitivity/specificity is not present in the table, the manufacturer needs to consider the 79 sample size associated with the largest sensitivity/specificity provided in the table that is smaller 80 to the claimed value (that is, as per the next smaller value of the sensitivity/ specificity available 81 in the table). For example, if a manufacturer claims a sensitivity of 93%, they are required to use 82 a sample size mentioned against 90% sensitivity.

f the sensitivity/ specificity available 81 in the table). For example, if a manufacturer claims a sensitivity of 93%, they are required to use 82 a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would 83 require usage of the sample size outlined for 85% specificity.
84 Positive samples: Positive samples should be positive by RT-PCR at least two ELISA kits from 85 the three mentioned above. If sufficient RT-PCR positive samples are not available, samples 86 positive by at least 2 ELISA kits (of the kits mentioned above) can be considered as true positive 87 samples. 88

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Negative samples: Samples which are negative by RT-PCR and at least two IgM ELISA kits 89 mentioned above will be considered as Chikungunya negative samples. 90 Table 1. Sample sizes and panel composition of positive chikungunya samples for different values 91 of sensitivity claimed by the manufacturer 92 Sensitivity Calculated sample size No.

s. 90 Table 1. Sample sizes and panel composition of positive chikungunya samples for different values 91 of sensitivity claimed by the manufacturer 92 Sensitivity Calculated sample size No. of Positive Samples required [Sample size rounded off] Sample Panel Composition 99%# 15 20 Strong positive: 4 Moderate positive: 8 Weak positive: 8 95% 73 80 Strong positive: 18 Moderate positive: 31 Weak positive: 31 90% 138 140 Strong positive: 30 Moderate positive: 55 Weak positive: 55 85% 196 200 Strong positive: 42 Moderate positive: 79 Weak positive: 79 80% 246 250 Strong positive: 54 Moderate positive: 98 Weak positive: 98 The samples need to be classified as strong, moderate and weak positives based on ELISA units of the reference assay.

#Higher sample size should be used even for assays claiming 99% sensitivity.

93 Table 2. Sample sizes and panel composition of negative chikungunya samples for different values 94 of specificity claimed by the manufacturer. 95 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off] 99%# 15 20 Rubella IgM positive: 1 Dengue IgM positive: 3 aAcute febrile illness cases: 8 bHealthy subjects from endemic regions: 8
95% 73 80 Rubella IgM positive: 5 Dengue IgM positive: 15 aAcute febrile illness cases: 30

Dengue IgM positive: 3 aAcute febrile illness cases: 8 bHealthy subjects from endemic regions: 8
95% 73 80 Rubella IgM positive: 5 Dengue IgM positive: 15 aAcute febrile illness cases: 30

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bHealthy subjects from endemic regions: 30 90% 138 140 Rubella IgM positive: 8 Dengue IgM positive: 26 aAcute febrile illness cases: 53 bHealthy subjects from endemic regions: 53 85% 196 200 Rubella IgM positive: 12 Dengue IgM positive: 38 aAcute febrile illness cases: 75 bHealthy subjects from endemic regions: 75 80% 246 250 Rubella IgM positive: 15 Dengue IgM positive: 47 aAcute febrile illness cases: 94 bHealthy subjects from endemic regions: 94 a Acute febrile illness cases negative for above pathogens AND Chikungunya IgM & PCR b Samples from healthy subjects from endemic regions negative for all Chikungunya markers (IgM, RNA) #Higher sample size should be used even for assays claiming 99% specificity.

96 6. Test reproducibility 97 A. Sample size for lot-to-lot reproducibility 98 Three lots of an assay shall be evaluated. Sample size for lot-to-lot reproducibility should be 99 as follows:
100  First lot of the assay: should be tested on statistically significant number of positive 101 and negative samples as calculated in the protocol.
102  Second lot of the assay: should be tested on 25 samples (15 positive samples 103 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 104 samples).

protocol.
102  Second lot of the assay: should be tested on 25 samples (15 positive samples 103 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 104 samples).
105  Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 106 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 107

108 Refer the flowchart below (Fig.1): 109

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112 7. Acceptance Criteria 113 Expected sensitivity: ≥90% 114 Expected specificity: ≥95% 115 8. Publication Rights: 116 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead author(s). 117

118 After following due procedure as defined in this document, once any kit is found to be Not 119 of Standard Quality, thereafter, no request for repeat testing of the same kit will be 120 acceptable. Any request of re-validation from the same manufacturer for the same test type 121 will only be entertained if valid proof of change in the kit composition is submitted. 122

123 VI. References:
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1. Kikuti M, Tauro LB, Moreira PSS, et al. Evaluation of two commercially available Chikungunya 125 virus IgM enzyme-linked immunoassays (ELISA) in a setting of concomitant transmission of 126 Chikungunya, Dengue and Zika viruses. Int J Infect Dis. 2020 Feb;91:38-43. 127

lable Chikungunya 125 virus IgM enzyme-linked immunoassays (ELISA) in a setting of concomitant transmission of 126 Chikungunya, Dengue and Zika viruses. Int J Infect Dis. 2020 Feb;91:38-43. 127

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2. Johnson BW, Goodman CH, Holloway K, de Salazar PM, Valadere AM, Drebot MA. Evaluation of 128 Commercially Available Chikungunya Virus Immunoglobulin M Detection Assays. Am J Trop 129 Med Hyg. 2016 Jul 6;95(1):182-192. doi: 10.4269/ajtmh.16-0013. Epub 2016 Mar 14.
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3. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 131 Diagnostic Assessment TGS-3.

Available at: 132 https://iris.who.int/bitstream/handle/10665/258985/WHO-EMP-RHT-PQT-TGS3-2017.03- 133 eng.pdf;sequence=1
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PERFORMANCE EVALUATION REPORT FOR CHIKUNGUNYA IgM ELISA KIT 159

160 Name of the product (Brand /generic) Name and address of the legal manufacturer Name and address of the actual manufacturing site Name and address of the Importer Name of supplier: Manufacturer/Importer/Port office of CDSCO/State licensing Authority Lot No / Batch No.: Product Reference No/ Catalogue No Type of Assay Kit components Manufacturing Date Expiry Date Pack size (Number of tests per kit) Intended Use Number of Tests Received Regulatory Approval: Import license / Manufacturing license/ Test license

License Number: Issue date:

Valid Up to: Application No. Sample Panel Positive samples (provide details: strong, moderate, weak)
Negative samples (provide detail: clinical/spiked, including cross reactivity panel)

161 Results:
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Reference assay ……….……………… (name)

Positive Negative Total Name of Chikungunya antibody -based ELISA kit Positive

Negative

Total

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Estimate (%) 95% CI Sensitivity

Specificity

Conclusions: 164 o Sensitivity, specificity 165 o Performance: Satisfactory / Not satisfactory 166 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 167

ty, specificity 165 o Performance: Satisfactory / Not satisfactory 166 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 167 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 168

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169 Disclaimers 170

1. This validation process does not approve / disapprove the kit design 171
2. This validation process does not certify user friendliness of the kit / assay 172 Note: This report is exclusively for ………………………Kit (Lot No……) manufactured by …………… 173 (Supplied by ……….) 174 Evaluation Done on …………………… 175 Evaluation Done by …………………………. 176 Signature of Director/ Director-In-charge …………………… Seal ………………………… 177 ****End of the Report 178

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Performance evaluation protocol for Chikungunya IgM RDT kits 198 I. Background:
199 200 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish 201 202 203 II. Purpose: 204 To evaluate the performance characteristics of Chikungunya IgM RDT kits in the diagnosis of 205 Chikungunya infection. 206 III. Requirements:
207 208 209 210 211 212 213 214 IV. Ethical approvals:
215 216 217 218 V. Procedure: 219

1. Study design/type: Diagnostic accuracy study using archived/leftover clinical samples. 220 221 222 A.Accreditation form NABL for at least one of the Quality management system (NABL 223 224 225 B.Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 226 227 228 ⮚ Handling of Chikungunya IgM RDT kits received for performance evaluation 229 230

B.Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 226 227 228 ⮚ Handling of Chikungunya IgM RDT kits received for performance evaluation 229 230

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231 232 3. Preparation of Chikungunya IgM Rapid IVD kit evaluation panel 233 Well characterised Chikungunya IVD kit evaluation panel is a critical requirement for performance 234 evaluation of IVD kits. Hence statistically significant number of sera samples should be available 235 from Chikungunya confirmed cases. Further characterised for Chikungunya IgM positivity by 236 using approved reference kits having high sensitivity and specificity. 237 Chikungunya IgM performance evaluation panel need to be tested again by the reference assays at 238 the time of evaluating a particular index test to confirm the positive and negative status of the 239 samples. 240 4. Reference assay:
241 All the samples will be tested by CDC/NIV real-time PCR assay. Samples which are positive by 242 RT-PCR assay will be further tested by any two of the following IgM ELISA kits: 243 i. ICMR-NIV MAC ELISA kit 244 ii. Inbios CHIKjj Detect™ IgM ELISA 245 iii. Anti-Chikungunya virus ELISA (IgM) Test (Euroimmun, Luebeck, Germany) 246 Samples positive by at least two kits will be considered. If sufficient RT-PCR positive samples 247 are not available, samples positive by at least 2 ELISA kits (of the kits mentioned above) can 248 be considered as true positive samples.

be considered. If sufficient RT-PCR positive samples 247 are not available, samples positive by at least 2 ELISA kits (of the kits mentioned above) can 248 be considered as true positive samples.
249 Samples which are negative by RT-PCR and at least two IgM ELISA kits mentioned above will be 250 considered as Chikungunya negative samples. 251 5. Sample size and sample panel composition: Sample sizes of positive and negative 252 samples and sample panel composition against different values of sensitivity and specificity are 253 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of significance, 254 an absolute precision of 5%, and invalid test rate ≤5%. Appropriate sample size has to be chosen 255 from the tables according to the values of sensitivity and specificity being claimed by the 256 manufacturer. If a claimed sensitivity/specificity is not present in the table, the manufacturer needs 257 to consider the sample size associated with the largest sensitivity/specificity provided in the table 258 that is smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ 259 specificity available in the table). For example, if a manufacturer claims a sensitivity of 93%, they 260 are required to use a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% 261 specificity would require usage of the sample size outlined for 85% specificity.
262 Positive samples: Positive samples should be positive by RT-PCR at least two ELISA kits from 263 the three mentioned above.

uld require usage of the sample size outlined for 85% specificity.
262 Positive samples: Positive samples should be positive by RT-PCR at least two ELISA kits from 263 the three mentioned above. If sufficient RT-PCR positive samples are not available, samples 264 positive by at least 2 ELISA kits (of the kits mentioned above) can be considered as true positive 265 samples. 266

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Negative samples: Samples which are negative by RT-PCR and at least two IgM ELISA kits 267 mentioned above will be considered as Chikungunya negative samples. 268 Table 1. Sample sizes and panel composition of positive chikungunya samples for different values 269 of sensitivity claimed by the manufacturer. 270 Sensitivity Calculated sample size No. of Positive Samples required off] 99%# 16 20 Strong Positive: 6 Moderate Positive: 8 Weak Positive: 6 95% 77 80 Strong Positive: 23 Moderate Positive: 34 Weak Positive: 23 90% 145 150 Strong Positive: 43 Moderate Positive: 64 Weak Positive: 43 85% 206 210 Strong Positive: 61 Moderate Positive: 88 Weak Positive: 61 80% 258 260 Strong Positive: 75 Moderate Positive: 110 Weak Positive: 75 The samples need to be classified as strong, moderate and weak positives based on ELISA units of the reference assay.

#Higher sample size should be used even for assays claiming 99% sensitivity.

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272 Table 2. Sample sizes and panel composition of negative chikungunya samples for different values 273 of specificity claimed by the manufacturer.

for assays claiming 99% sensitivity.

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272 Table 2. Sample sizes and panel composition of negative chikungunya samples for different values 273 of specificity claimed by the manufacturer. 274 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off] 99%# 16 20 Rubella IgM positive: 1 Dengue IgM positive: 3 aAcute febrile illness cases: 12 bHealthy subjects from endemic regions: 4
95% 77 80 Rubella IgM positive: 3

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Dengue IgM positive: 13 aAcute febrile illness cases: 48 bHealthy subjects from endemic regions: 16 90% 145 150 Rubella IgM positive: 5 Dengue IgM positive: 25 aAcute febrile illness cases: 90 bHealthy subjects from endemic regions: 30 85% 206 210 Rubella IgM positive: 7 Dengue IgM positive: 35 aAcute febrile illness cases: 126 bHealthy subjects from endemic regions: 42 80% 258 260 Rubella IgM positive: 9 Dengue IgM positive: 43 aAcute febrile illness cases: 156 bHealthy subjects from endemic regions: 52 a Acute febrile illness cases negative for above pathogens AND Chikungunya IgM & PCR b Samples from healthy subjects from endemic regions negative for all Chikungunya markers (IgM, RNA)

#Higher sample size should be used even for assays claiming 99% specificity.

275 6. Test reproducibility 276 277 278 as follows:
279 280 281 282 283 samples).
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287 Refer the flowchart below (Fig. 1): 288

for assays claiming 99% specificity.

275 6. Test reproducibility 276 277 278 as follows:
279 280 281 282 283 samples).
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287 Refer the flowchart below (Fig. 1): 288

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291 B. Sample size for reader-to-reader reproducibility
292 For reader-to-reader reproducibility, sample size should be 25 (15 positive samples 293 294 samples). 295

296 Two operators will be reading the test results independently as per manufacturer’s instruction. 297 Agreement should be 100% between the operators. 298 7. Acceptance criteria 299 Expected sensitivity: ≥80% 300 Expected specificity: ≥90% 301 Invalid test rate: ≤5% 302 8. Publication Rights: 303 304

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308 309 VI. References: 310

1. Kikuti M, Tauro LB, Moreira PSS, et al. Evaluation of two commercially available 311 Chikungunya virus IgM enzyme-linked immunoassays (ELISA) in a setting of 312 concomitant transmission of Chikungunya, Dengue and Zika viruses. Int J Infect Dis. 313 2020 Feb;91:38-43. 314
2. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 315 Diagnostic Assessment TGS-3.

Available at: 316 317 eng.pdf;sequence=1
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PERFORMANCE EVALUATION REPORT FOR CHIKUNGUNYA IgM RDT KIT 340 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: strong, moderate, weak)
Negative samples (provide details: clinical/spiked, including cross reactivity panel) Results:
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Positive Negative Total Name of Chikungunya antibody - based RDT kit Positive

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Estimate (%) 95% CI Sensitivity

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Conclusions: 343 o Sensitivity, specificity 344 o Performance: Satisfactory / Not satisfactory 345 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 346 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 347

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sessed in controlled lab setting using kits provided by the manufacturer from 346 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 347

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349 Disclaimers 350 351 352 Note: This report is exclusively for ………………………Kit (Lot No……) manufactured by …………… 353 (Supplied by ……….) 354 355 356 Signature of Director/ Director-In-charge …………………… Seal …………………….. 357

358 ****End of the Report 359

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Performance evaluation protocol for Chikungunya real-time PCR kits 379 I. Background:
380 381 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish the 382 383 384 II. Purpose: 385 To evaluate the performance characteristics of Chikungunya PCR kits in the diagnosis of 386 Chikungunya infection. 387 III. Requirements:
388 389 390 391 392 393 394 395 IV. Ethical approvals:
396 397 398 399 V. Procedure: 400

1. Study design/type: Diagnostic accuracy study using archived/ leftover/spiked clinical 401 samples. 402 403 404 405 406 407 408 409 410 ⮚ Handling of Chikungunya PCR kits received for performance evaluation 411 412

y study using archived/ leftover/spiked clinical 401 samples. 402 403 404 405 406 407 408 409 410 ⮚ Handling of Chikungunya PCR kits received for performance evaluation 411 412

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413 414 3. Preparation of Chikungunya RNA evaluation panel 415 Well characterised Chikungunya sample panel positive for RNA is a critical requirement for 416 performance evaluation of IVD kits utilizing genome detection. Hence statistically significant 417 number of sera/whole blood samples should be available from Chikungunya PCR confirmed cases.
418 4. RNA extraction 419 RNA extraction should be performed using a standard RNA extraction kit using spin columns such 420 as QIAamp Viral RNA Mini kitor MDI Viral Mini RNA Extraction Mini Prep Kit or magnetic 421 bead-based extraction methods such as MagMax viral RNA isolation kit. 422 If the manufacturer of the index test recommends a specific RNA extraction kit, the same needs to 423 be provided by the manufacturer. 424 5. Real-Time PCR System 425 PCR shall be performed using IVD-approved machines. If any equipment(s) is specified in the 426 IFU of the index test, it shall be used for the evaluation, and it shall be provided by the 427 manufacturer if not available within the lab’s IVD evaluation scope. 428 6. Internal control/Extraction control 429 The test under evaluation should have an internal control or extraction control (RNA added before 430 extraction to a sample).
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on scope. 428 6. Internal control/Extraction control 429 The test under evaluation should have an internal control or extraction control (RNA added before 430 extraction to a sample).
431 7. Reference assay:
432 Any FDA approved Chikungunya PCR assay or CDC/NIV protocol for detection of Chikungunya 433 RNA should be used as the reference assay. 434 All positive samples should be confirmed positive for Chikungunya by reference assay. 435 All negative samples should be negative for all markers of Chikungunya infection (RNA using 436 reference assay AND IgM using any two of the following kits - ICMR-NIV MAC ELISA 437 kit/Inbios CHIKjj Detect™ IgM ELISA/Anti-Chikungunya virus ELISA (IgM) Test (Euroimmun, 438 Luebeck, Germany). 439 8. Sample size and sample panel composition: Sample sizes of positive and negative 440 samples and sample panel composition against different values of sensitivity and specificity are 441 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of significance, 442 an absolute precision of 5%, and invalid test rate ≤5%. Appropriate sample size has to be chosen 443 from the tables according to the values of sensitivity and specificity being claimed by the 444 manufacturer. If a claimed sensitivity/specificity is not present in the table, the manufacturer needs 445 to consider the sample size associated with the largest sensitivity/specificity provided in the table 446

a claimed sensitivity/specificity is not present in the table, the manufacturer needs 445 to consider the sample size associated with the largest sensitivity/specificity provided in the table 446

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that is smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ specificity 447 available in the table). For example, if a manufacturer claims a sensitivity of 93%, they are required 448 to use a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would 449 require usage of the sample size outlined for 85% specificity.
450 Table 1. Sample sizes and panel composition of positive chikungunya samples for different values 451 of sensitivity claimed by the manufacturer. 452 Sensitivity Calculated sample size No. of Positive Samples required off] 99%# 16 20 Strong Positive: 5 Moderate Positive: 10 Weak Positive: 5 95% 77 80 Strong Positive: 20 Moderate Positive: 40 Weak Positive: 20 90% 145 150 Strong Positive: 38 Moderate Positive: 74 Weak Positive: 38 85% 206 210 Strong Positive: 53 Moderate Positive: 104 Weak Positive: 53 #Higher sample size should be used even for assays claiming 99% sensitivity. 453 Strong positive (Ct value between <25)
454 Moderate positive (Ct value between 25-30)
455 Weak positive (Ct value between >30 to 34)
456

457 Table 2. Sample sizes and panel composition of negative chikungunya samples for different values 458 of specificity claimed by the manufacturer.

positive (Ct value between >30 to 34)
456

457 Table 2. Sample sizes and panel composition of negative chikungunya samples for different values 458 of specificity claimed by the manufacturer. 459 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off] 99%# 16 20 Rubella IgM positive: 1 Dengue IgM positive: 4 aAcute febrile illness cases: 10 bHealthy subjects from endemic regions: 5
95% 77 80 Rubella IgM positive: 5

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 21 | Page

Dengue IgM positive: 15 aAcute febrile illness cases: 40 bHealthy subjects from endemic regions: 20 90% 145 150 Rubella IgM positive: 9 Dengue IgM positive: 28 aAcute febrile illness cases: 75 bHealthy subjects from endemic regions: 38 85% 206 210 Rubella IgM positive: 13 Dengue IgM positive: 39 aAcute febrile illness cases: 105 bHealthy subjects from endemic regions: 53 a Acute febrile illness cases negative for above pathogens AND Chikungunya IgM & PCR b Samples from healthy subjects from endemic regions negative for all Chikungunya markers (IgM, RNA) #Higher sample size should be used even for assays claiming 99% specificity. 460 9. Evaluation method: 461 The index test and the reference tests should be run simultaneously on the sample panel to avoid 462 false negative results by index test due to free thawing of samples or deterioration of sample quality 463 on long term storage. 464 10. Test reproducibility 465 466 467 as follows:
468 469 470 471 472 samples).
473 474 475 476

thawing of samples or deterioration of sample quality 463 on long term storage. 464 10. Test reproducibility 465 466 467 as follows:
468 469 470 471 472 samples).
473 474 475 476

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 22 | Page

477

478 11. Acceptance criteria 479 Expected sensitivity: ≥95% 480 Expected specificity: ≥98% 481 Cross reactivity with related viruses: NIL 482 483 11. Publication Rights: 484 485 486 487 488 489

490 VI. References: 491

1. Santiago, G.A., Vázquez, J., Courtney, S. et al. Performance of the Trioplex real-time RT-PCR assay 492 for detection of Zika, Dengue, and Chikungunya viruses. Nat Commun 9, 1391 (2018). 493 https://doi.org/10.1038/s41467-018-03772-1 494
2. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 495 Diagnostic Assessment TGS-3.

Available at: 496

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 23 | Page

497 eng.pdf;sequence=1
498

499 VII. 500

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ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 24 | Page

PERFORMANCE EVALUATION REPORT FOR CHIKUNGUNYA REAL-TIME PCR 526 KITS 527 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: clinical/spiked, strong, moderate, weak)
Negative samples (provide details: clinical/spiked, including cross reactivity panel)

528 Results 529

Positive Negative Total Name of Chikungunya real-time PCR kits Positive

Negative

Total

530

Estimate (%) 95% CI Sensitivity

Specificity

531 ● Conclusions: 532 o Cross reactivity with related viruses: 533 o Performance: Satisfactory / Not satisfactory 534

530

Estimate (%) 95% CI Sensitivity

Specificity

531 ● Conclusions: 532 o Cross reactivity with related viruses: 533 o Performance: Satisfactory / Not satisfactory 534

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 25 | Page

(Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from the batch 535 mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 536

537 Disclaimers 538 539 540 Note: This report is exclusively for Chikungunya………….. Kit (Lot No……) manufactured by …………… 541 (supplied by ……….) 542 543 544 Signature of Director/ Director-In-charge …………………… Seal ………………………….. 545 ****End of the Report 546

547

548

549

550

551

552

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560

561

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563

564

565

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 26 | Page

Performance evaluation protocol for Dengue NS1 RDT kits 566 I. Background:
567 568 569 570 evaluation is to independently verify the manufacturer’s claim IVD performance. 571 II. Purpose: 572 To evaluate the performance characteristics of Dengue NS1 RDT kits in the diagnosis of Dengue 573 infection. 574 III. Requirements:
575 576 577 578 579 580 581 582 IV. Ethical approvals:
583 584 585 586 V. Procedure: 587

1. Study design/type: Diagnostic accuracy study using archived/leftover clinical samples.

575 576 577 578 579 580 581 582 IV. Ethical approvals:
583 584 585 586 V. Procedure: 587

1. Study design/type: Diagnostic accuracy study using archived/leftover clinical samples. 588 589 590 591 592 593 594 595 596 ⮚ Handling of Dengue NS1 Rapid IVD kits received for performance evaluation 597 598

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 27 | Page

599 600 3. Preparation of Dengue RDT IVD kit evaluation panel 601 Well characterised Dengue NS1 RDT IVD kit evaluation panel is a critical requirement for 602 performance evaluation of IVD kits. Hence statistically significant number of sera samples should 603 be available from Dengue confirmed cases. Further characterised for Dengue NS1 positivity by 604 using approved reference kits having high sensitivity and specificity. 605 Dengue NS1 performance evaluation panel need to be tested again by the reference assays at the 606 time of evaluating a particular index test to confirm the positive and negative status of the samples. 607 4. Reference assay:
608 US-FDA approved Dengue NS1 ELISA kit should be used as reference assay. 609 Serotype status to be assessed using CDC/NIV real-time PCR serotyping protocols.
610 5. Sample size and sample panel composition: Sample sizes of positive and negative 611 samples and sample panel composition against different values of sensitivity and specificity are 612 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of significance, 613 an absolute precision of 5%, and invalid test rate ≤5%.

f sensitivity and specificity are 612 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of significance, 613 an absolute precision of 5%, and invalid test rate ≤5%. Appropriate sample size has to be chosen 614 from the tables according to the values of sensitivity and specificity being claimed by the 615 manufacturer. If a claimed sensitivity/specificity is not present in the table, the manufacturer needs 616 to consider the sample size associated with the largest sensitivity/specificity provided in the table 617 that is smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ 618 specificity available in the table). For example, if a manufacturer claims a sensitivity of 93%, they 619 are required to use a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% 620 specificity would require usage of the sample size outlined for 85% specificity.
621 Positive samples: The panel of positive samples should include samples positive by the reference 622 assay and real-time PCR assay (True positives). Samples should be representative of all 4 serotypes 623 and varying degrees of positivity. The samples should be classified as strong, moderate and weak 624 positives based on ELISA units of the reference assay. 625

626 Negative samples: These should include samples negative by the reference NS1 ELISA assay and 627 real-time PCR using CDC/NIV serotyping protocol (True negatives). 628 Table 1.

assay. 625

626 Negative samples: These should include samples negative by the reference NS1 ELISA assay and 627 real-time PCR using CDC/NIV serotyping protocol (True negatives). 628 Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 629 sensitivity claimed by the manufacturer. 630 Sensitivity Calculated sample size No. of Positive Samples required off]

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 28 | Page

99%# 16 20 Samples should be representative of all 4 serotypes and varying degrees of positivity, with at least 25% weak positive samples. 95% 77 80 90% 145 150 85% 206 210 80% 258 260

631 #Higher sample size should be used even for assays claiming 99% sensitivity. 632 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 633 specificity claimed by the manufacturer. 634 Specificity Calcula ted sample size No. of Negative Samples required [Sample size rounded off] 99%# 16 20 -PCR/RT-PCR positive samples from other acute febrile illness cases Chikungunya: 4 Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR): 8

-Samples from other flavivirus disease cases *Japanese Encephalitis PCR/antigen positive: 1 *West Nile Virus PCR/antigen positive: 1 *Zika Virus PCR/antigen positive: 1

-Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid): 5

95% 77 80 -PCR/RT-PCR positive samples from other acute febrile illness cases Chikungunya: 15

ubjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid): 5

95% 77 80 -PCR/RT-PCR positive samples from other acute febrile illness cases Chikungunya: 15 Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR): 30

-Samples from other flavivirus disease cases *Japanese Encephalitis PCR/antigen positive: 5 *West Nile Virus PCR/antigen positive: 5 *Zika Virus PCR/antigen positive: 5

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 29 | Page

-Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid): 20

90% 145 150 -PCR/RT-PCR positive samples from other acute febrile illness cases Chikungunya: 28 Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR): 57

-Samples from other flavivirus disease cases *Japanese Encephalitis PCR/antigen positive: 9 *West Nile Virus PCR/antigen positive: 9 *Zika Virus PCR/antigen positive: 9

-Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid): 38

85% 206 210 -PCR/RT-PCR positive samples from other acute febrile illness cases Chikungunya: 39 Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR): 79

-Samples from other flavivirus disease cases *Japanese Encephalitis PCR/antigen positive: 13 *West Nile Virus PCR/antigen positive 13 *Zika Virus PCR/antigen positive: 13

-Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid): 53

80% 258 260

/antigen positive 13 *Zika Virus PCR/antigen positive: 13

-Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid): 53

80% 258 260 -PCR/RT-PCR positive samples from other acute febrile illness cases Chikungunya: 49 Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR): 98

-Samples from other flavivirus disease cases *Japanese Encephalitis PCR/antigen positive: 16

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 30 | Page

*West Nile Virus PCR/antigen positive: 16 *Zika Virus PCR/antigen positive: 16

-Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid): 65

• In the absence of natural samples, spiked samples may be used, as per details provided in the note below.

Recombinant NS1 antigen of cross reactive flaviviruses (Zika, West Nile and Japanese Encephalitis viruses) expressed in mammalian cells can be obtained commercially and reconstituted in serum samples (100 ng -1 µg/ml) and diluted in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1). Before used for evaluation, flavivirus NS1 reconstituted in serum samples needs to be tested by the dengue NS1 reference assay, and dilutions which are negative for dengue should be used for evaluation. The serum samples used for reconstitution should be negative for Dengue NS1, RNA and IgM antibody.

#Higher sample size should be used even for assays claiming 99% specificity.

635 6.

ation. The serum samples used for reconstitution should be negative for Dengue NS1, RNA and IgM antibody.

#Higher sample size should be used even for assays claiming 99% specificity.

635 6. Test reproducibility 636 637 638 as follows:
639 640 641 642 643 samples).
644 645 646

647 648

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 31 | Page

649

650 B. Sample size for reader-to-reader reproducibility 651 For reader-to-reader reproducibility, sample size should be 25 (15 positive samples comprising 10 652 low positive AND 5 moderate/high positive samples, and 10 negative samples). 653

654 Two operators will be reading the test results independently as per manufacturer’s instruction. 655 Agreement should be 100% between the operators. 656 7. Criteria for approval of the Dengue NS1 RDT kits 657 Expected sensitivity: ≥80% 658 Expected specificity: ≥95% 659 Cross reactivity with other flavivirus antigens: Nil 660 661 9. Publication Rights: 662 663

664 665 666

DT kits 657 Expected sensitivity: ≥80% 658 Expected specificity: ≥95% 659 Cross reactivity with other flavivirus antigens: Nil 660 661 9. Publication Rights: 662 663

664 665 666

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 32 | Page

667 668

669 VI. References: 670

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian 671 E, Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, 672 Peeling RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection 673 of Dengue virus NS1 antigen and anti-Dengue virus IgM antibody. PLoSNegl Trop Dis. 2014 Oct 674 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 675
2. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, 676 Nisalak A, Yoon IK, Fernandez S. Evaluation of a Dengue NS1 antigen detection assay 677 sensitivity and specificity for the diagnosis of acute Dengue virus infection. PLoSNegl 678 Trop Dis. 2014 Oct 2;8(10):e3193. doi: 10.1371/journal.pntd.0003193. 679
3. Yow KS, Aik J, Tan EY, Ng LC, Lai YL. Rapid diagnostic tests for the detection of recent 680 Dengue infections: An evaluation of six kits on clinical specimens. PLoS One. 2021 Apr 681 1;16(4): e0249602. doi: 10.1371/journal.pone.0249602. 682
4. Mat Jusoh TNA, Shueb RH. Performance Evaluation of Commercial Dengue Diagnostic 683 Tests for Early Detection of Dengue in Clinical Samples. J Trop Med. 2017; 2017: 684
5. doi: 10.1155/2017/4687182. Epub 2017 Dec 12.

rformance Evaluation of Commercial Dengue Diagnostic 683 Tests for Early Detection of Dengue in Clinical Samples. J Trop Med. 2017; 2017: 684 4687182. doi: 10.1155/2017/4687182. Epub 2017 Dec 12. PMID: 29379526; PMCID: 685 PMC5742879. 686 5. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 687 Diagnostic Assessment TGS-3. 2017. Available at: 688 689 eng.pdf;sequence=1
690 6. Mahajan R, Nair M, Saldanha AM, Harshana A, Pereira AL, Basu N, Goswami RP, 691 Bhattacharya N, Bandyopadhay B, SenGupta M, Day M, Flevaud L, Boelaert M, 692 Burza S. Diagnostic accuracy of commercially available immunochromatographic 693 rapid tests for diagnosis of dengue in India. J Vector Borne Dis. 2021 Apr- 694 Jun;58(2):159-164. doi: 10.4103/0972-9062.321747. PMID: 35074951. 695

696 VII. 697

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ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 33 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE NS1 RDT KIT 706 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No.

Page

PERFORMANCE EVALUATION REPORT FOR DENGUE NS1 RDT KIT 706 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: clinical/spiked, strong, moderate, weak)
Negative samples (provide details: clinical/spiked, including cross reactivity panel)

707 Results: 708

Positive Negative Total Name of Dengue NS1 - based RDT kit Positive

Negative

Total

709

Estimate (%) 95% CI Sensitivity

Specificity

710 ● Details of cross reactivity with other flavivirus NS1 antigens:
711 ● Conclusions: 712 o Sensitivity, specificity 713 o 714

715

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 34 | Page

(Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from the batch 716 mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 717

718 Disclaimers 719 720 721

722 Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by …………… (Supplied by ……….) 723 724 725 Signature of Director/ Director-In-charge …………………… Seal ……………………….. 726 ****End of the Report 727

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End of the Report****** 727

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ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 35 | Page

Field evaluation protocol for Dengue NS1 RDT kits 746 I. Background:
747 748 749 750 751 II. Purpose: 752 To evaluate the performance characteristics of Dengue NS1 RDT kits in the diagnosis of Dengue 753 infection in individuals with unknown disease status. 754 III. Requirements:
755 756 757 758 759 760 4. Laboratory supplies
761

762 IV. Ethical approval:
763 The study will be initiated after approval from the institutional human ethics committee.
764 V. Procedure: 765

1. Study design/type: Cross-sectional study 766 767 768 769 770 771 772 773 774 ⮚ Handling of Dengue NS1 RDT IVD kits received for performance evaluation 775 776 777 778

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 36 | Page

3. Sample size for performance evaluation: 779 Sample sizes of positive and negative samples of Dengue against different values of 780 sensitivity and specificity are provided in Tables 1 and 2. Sample sizes have been calculated 781 assuming 95% level of significance, an absolute precision of 5%, and invalid test rate ≤5%. 782 It is further assumed that 30% of the individuals attending the health care facilities for acute 783 febrile illness and suspected for Dengue will be positive for Dengue.

valid test rate ≤5%. 782 It is further assumed that 30% of the individuals attending the health care facilities for acute 783 febrile illness and suspected for Dengue will be positive for Dengue. Appropriate sample 784 size has to be chosen from the tables according to the values of sensitivity and specificity 785 being claimed by the manufacturer. If a claimed sensitivity/specificity is not present in the 786 table, the manufacturer needs to consider the sample size associated with the largest 787 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as 788 per the next smaller value of the sensitivity/ specificity available in the table). For example, 789 if a manufacturer claims a sensitivity of 93%, they are required to use a sample size 790 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 791 usage of the sample size outlined for 85% specificity.
792 Sample size has to be calculated based on both the sensitivity and the specificity. The 793 final sample size will be the maximum of the two. For example, at 95% sensitivity and 794 95% specificity, the sample size required will be 260 (maximum of 260 and 110).
795

796 Table 1. Sample sizes for different values of sensitivity claimed by the manufacturer. 797 Sensitivity Calculated sample size No.

ize required will be 260 (maximum of 260 and 110).
795

796 Table 1. Sample sizes for different values of sensitivity claimed by the manufacturer. 797 Sensitivity Calculated sample size No. of individuals* off] 99%# 53 60 95% 255 260 90% 484 490 85% 686 690 80% 861 870

• Individuals attending the health care facilities for acute febrile illness and suspected for Dengue meeting the inclusion criteria

798 #Higher sample size should be used even for assays claiming 99% sensitivity. 799

800 Table 2. Sample sizes for different values of specificity claimed by the manufacturer. 801 Specificity Calculated sample size No. of individuals* off] 99%# 23 30 95% 109 110 90% 207 210 85% 294 300 80% 369 370

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 37 | Page

• Individuals attending the health care facilities for

802 #Higher sample size should be used even for assays claiming 99% specificity. 803 Recruitment of cases shall be halted once desired number of positive and negative samples are 804 reached. 805 4. Inclusion criteria: 806 Individuals with Dengue like illness (An individual with acute febrile illness of 2-7 days with two 807 or more manifestations: Head ache, retro-orbital pain, myalgia, arthralgia, rash, hemorrhagic 808 manifestations) 809 5. Exclusion criteria: 810 Individuals with already known positive history for other pathogens 811 6. Reference assay:
812 US-FDA approved Dengue NS1 ELISA kit should be used as reference assay.

Exclusion criteria: 810 Individuals with already known positive history for other pathogens 811 6. Reference assay:
812 US-FDA approved Dengue NS1 ELISA kit should be used as reference assay. 813 Serotype status to be assessed using CDC/NIV real-time PCR serotyping protocols.
814 7. Study implementation: 815 The individuals with Dengue like illness will be recruited into the study and five ml of whole blood 816 will be collected in vacutainer tubes and the serum will be separated by centrifugation and used 817 for the study. The serum sample will be subjected to the following reference tests and the index 818 test. 819 It needs to be ensured that the samples are tested by reference tests and index test simultaneously. 820 8. Positive samples: 821 Samples positive by the reference NS1 ELISA assay and real-time PCR assay will be considered 822 as true positive sample.
823 9. Negative samples:
824 Samples negative by the reference NS1 ELISA assay and real-time PCR using CDC/NIV 825 serotyping protocol will be considered as true negative. 826 A. Cross reactivity: 827 Clinical samples or commercially available NS1 antigens from other flaviviruses will be used to 828 test cross reactivity of the index test. 829 i. Japanese Encephalitis PCR/antigen positive: 5 samples* 830 ii. West Nile Virus PCR/antigen: 5 samples* 831

r flaviviruses will be used to 828 test cross reactivity of the index test. 829 i. Japanese Encephalitis PCR/antigen positive: 5 samples* 830 ii. West Nile Virus PCR/antigen: 5 samples* 831

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 38 | Page

iii. Zika Virus PCR/antigen: 5 samples* 832 *In the absence of natural samples, spiked samples may be used, as per details provided in the note below. 833 Note:
834 Recombinant NS1 antigen of cross reactive flaviviruses (Zika, West Nile and Japanese Encephalitis viruses) expressed 835 in mammalian cells can be obtained commercially and reconstituted in serum samples (100 ng -1 µg/ml) and diluted 836 in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1). 837 Before used for evaluation, flavivirus NS1 reconstituted in serum samples needs to be tested by the dengue NS1 838 reference assay, and dilutions which are negative for dengue should be used for evaluation. 839 The serum samples used for reconstitution should be negative for Dengue NS1, RNA and IgM antibody. 840 10. Statistical analysis:
841 Sensitivity and specificity will be calculated. 842 Interim analysis of data shall be conducted on completing evaluation of 25%, 50% and 75% of 843 samples. If, at any point, the performance of the assay is found to be not satisfactory, the assay 844 shall not be evaluated further. Evaluation fee shall be charged accordingly. 845 11. Test reproducibility 846 847 848 as follows:
849 850 851 852 853 samples).
854 855 856 857

844 shall not be evaluated further. Evaluation fee shall be charged accordingly. 845 11. Test reproducibility 846 847 848 as follows:
849 850 851 852 853 samples).
854 855 856 857

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 39 | Page

858 B. Sample size for reader-to-reader reproducibility 859 For reader-to-reader reproducibility, sample size should be 25 (15 positive samples comprising 10 860 low positive AND 5 moderate/high positive samples, and 10 negative samples). 861

862 Two operators will be reading the test results independently as per manufacturer’s instruction. 863 Agreement should be 100% between the operators. 864 12. Acceptance Criteria 865 Expected sensitivity: ≥80% 866 Expected specificity: ≥95% 867 Cross-reactivity with other flavivirus antigens: Nil 868 869 13. Publication Rights: 870 871

872 873 874 875 876

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 40 | Page

877 VI. References: 878

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian E, 879 Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, Peeling 880 RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection of 881 Dengue virus NS1 antigen and anti-Dengue virus IgM antibody. PLoSNegl Trop Dis. 2014 Oct 882 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 883
2. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, Nisalak A, Yoon 884 IK, Fernandez S.

rop Dis. 2014 Oct 882 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 883 2. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, Nisalak A, Yoon 884 IK, Fernandez S. Evaluation of a Dengue NS1 antigen detection assay sensitivity and specificity for 885 the diagnosis of acute Dengue virus infection. PLoSNegl Trop Dis. 2014 Oct 2;8(10):e3193. doi: 886 10.1371/journal.pntd.0003193. 887 3. Ganeshkumar P, Murhekar MV, Poornima V, Saravanakumar V, Sukumaran K, Anandaselvasankar 888 A, John D, Mehendale SM. Dengue infection in India: A systematic review and meta-analysis. 889 PLoSNegl Trop Dis. 2018 Jul 16;12(7):e0006618. doi: 10.1371/journal.pntd.0006618. 890

891 4. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 892 Diagnostic Assessment TGS-3. 2017. Available at: 893 894 eng.pdf;sequence=1
895 5. Mahajan R, Nair M, Saldanha AM, Harshana A, Pereira AL, Basu N, Goswami RP, Bhattacharya 896 N, Bandyopadhay B, SenGupta M, Day M, Flevaud L, Boelaert M, Burza S. Diagnostic accuracy 897 of commercially available immunochromatographic rapid tests for diagnosis of dengue in India. 898 J Vector Borne Dis. 2021 Apr-Jun;58(2):159-164. doi: 10.4103/0972-9062.321747. PMID: 899 35074951. 900 VII. 901

902

903

904

905

906

907

908

909

910

911

912

913

914

Borne Dis. 2021 Apr-Jun;58(2):159-164. doi: 10.4103/0972-9062.321747. PMID: 899 35074951. 900 VII. 901

902

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905

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ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 41 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE NS1 RDT KIT 915 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples: Not applicable, may categorize cases as per duration of illness Negative samples (may categorize as per duration of illness, must include cross reactivity panel)

916 Results: 917

Positive Negative Total Name of Dengue NS1 - based RDT kit Positive

Negative

Total

918

Estimate (%) 95% CI Sensitivity

Specificity

919

920 ● Details of cross reactivity with other flavivirus NS1 antigens:
921 ● Conclusions: 922 o Sensitivity, specificity 923 o 924

(%) 95% CI Sensitivity

Specificity

919

920 ● Details of cross reactivity with other flavivirus NS1 antigens:
921 ● Conclusions: 922 o Sensitivity, specificity 923 o 924

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 42 | Page

(Sensitivity and specificity have been assessed in using kits provided by the manufacturer from the batch mentioned above using 925 ….. sample in ……. (field/controlled lab). Results should not be extrapolated to other sample types.) 926

927 Disclaimers 928 929 930

931 Note: This report is exclusively for NS1…………………Kit (Lot No……) manufactured by …………… (supplied 932 by ……….) 933 934 935 Signature of Director/ Director-In charge …………………… Seal …………………………….. 936 ****End of the Report 937

938

939

940

941

942

943

944

945

946

947

948

949

950

951

952

953

954

955

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 43 | Page

Performance evaluation protocol for Dengue NS1 ELISA kits 956 I. Background:
957 958 959 960 961 II. Purpose: 962 To evaluate the performance characteristics of Dengue NS1 ELISA kits in the diagnosis of Dengue 963 infection. 964 III. Requirements:
965 966 967 968 969 970 971 972 IV. Ethical approvals:
973 974 975 976 V. Procedure: 977

1. Study design/type: Diagnostic accuracy study using archived/leftover clinical samples. 978 979 980 981 982 983 984 985 986 ⮚ Handling of Dengue NS1 ELISA kits received for performance evaluation 987 988

e: Diagnostic accuracy study using archived/leftover clinical samples. 978 979 980 981 982 983 984 985 986 ⮚ Handling of Dengue NS1 ELISA kits received for performance evaluation 987 988

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 44 | Page

989 990 3. Preparation of Dengue NS1 ELISA IVD kit evaluation panel 991 Well characterised Dengue NS1 ELISA IVD kit evaluation panel is a critical requirement for 992 performance evaluation of IVD kits. Hence statistically significant number of sera samples should 993 be available from Dengue confirmed cases. Further characterised for Dengue NS1 positivity by 994 using approved reference kits having high sensitivity and specificity. 995 Dengue NS1 performance evaluation panel need to be tested again by the reference assays at the 996 time of evaluating a particular index test to confirm the positive and negative status of the samples. 997 4. Reference assay:
998 US-FDA approved Dengue NS1 ELISA kit should be used as reference assay. 999 Serotype status to be assessed using CDC/NIV real-time PCR serotyping protocols.
1000 5. Sample size and sample panel composition: Sample sizes of positive and negative 1001 samples and sample panel composition against different values of sensitivity and specificity are 1002 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of significance, 1003 and an absolute precision of 5%. Appropriate sample size has to be chosen from the tables according 1004 to the values of sensitivity and specificity being claimed by the manufacturer.

ance, 1003 and an absolute precision of 5%. Appropriate sample size has to be chosen from the tables according 1004 to the values of sensitivity and specificity being claimed by the manufacturer. If a claimed 1005 sensitivity/specificity is not present in the table, the manufacturer needs to consider the sample size 1006 associated with the largest sensitivity/specificity provided in the table that is smaller to the claimed 1007 value (that is, as per the next smaller value of the sensitivity/ specificity available in the table). For 1008 example, if a manufacturer claims a sensitivity of 93%, they are required to use a sample size 1009 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require usage of the 1010 sample size outlined for 85% specificity.
1011 Positive samples: The panel of positive samples should include samples positive by the reference 1012 assay and real-time PCR assay (True positives). Samples should be representative of all 4 serotypes 1013 and varying degrees of positivity. The samples should be classified as strong, moderate and weak 1014 positives based on ELISA units of the reference assay. 1015

1016 Negative samples: These should include samples negative by the reference NS1 ELISA assay and 1017 real-time PCR using CDC/NIV serotyping protocol (True negatives). 1018

1019 Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 1020 sensitivity claimed by the manufacturer. 1021 Sensitivity Calculated sample size No.

1019 Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 1020 sensitivity claimed by the manufacturer. 1021 Sensitivity Calculated sample size No. of Positive Samples required

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 45 | Page

off] 99%# 15 20 Strong Positive: 4 Moderate Positive: 8 Weak Positive: 8 95% 73 80 Strong Positive: 18 Moderate Positive: 31 Weak Positive: 31 90% 138 140 Strong Positive: 30 Moderate Positive: 55 Weak Positive: 55 85% 196 200 Strong Positive: 42 Moderate Positive: 79 Weak Positive: 79 80% 246 250 Strong Positive: 54 Moderate Positive: 98 Weak Positive: 98

1022 #Higher sample size should be used even for assays claiming 99% sensitivity. 1023 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 1024 specificity claimed by the manufacturer. 1025 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off] 99%# 15 20 Chikungunya positive: 4
aAcute febrile cases negative for Dengue: 8
*Japanese Encephalitis PCR/antigen positive: 1
*West Nile Virus PCR/antigen positive: 1
*Zika Virus PCR/antigen positive: 1 bHealthy subjects from endemic regions: 5
95% 73 80 Chikungunya positive: 15
aAcute febrile cases negative for Dengue: 30
*Japanese Encephalitis PCR/antigen positive: 5
*West Nile Virus PCR/antigen positive: 5
*Zika Virus PCR/antigen positive: 5 bHealthy subjects from endemic regions: 20 90% 138 140 Chikungunya positive: 26

halitis PCR/antigen positive: 5
*West Nile Virus PCR/antigen positive: 5
*Zika Virus PCR/antigen positive: 5 bHealthy subjects from endemic regions: 20 90% 138 140 Chikungunya positive: 26
aAcute febrile cases negative for Dengue: 52
*Japanese Encephalitis PCR/antigen positive: 9

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 46 | Page

*West Nile Virus PCR/antigen positive: 9
*Zika Virus PCR/antigen positive: 9 bHealthy subjects from endemic regions: 35 85% 196 200 Chikungunya positive: 37
aAcute febrile cases negative for Dengue: 74
*Japanese Encephalitis PCR/antigen positive: 13
*West Nile Virus PCR/antigen positive:13
*Zika Virus PCR/antigen positive: 13 bHealthy subjects from endemic regions: 50 80% 246 250 Chikungunya positive: 46
aAcute febrile cases negative for Dengue: 94
*Japanese Encephalitis PCR/antigen positive: 16
*West Nile Virus PCR/antigen positive: 16
*Zika Virus PCR/antigen positive: 16 bHealthy subjects from endemic regions: 62 a Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid)

#Higher sample size should be used even for assays claiming 99% specificity.

1026 *In the absence of natural samples, spiked samples may be used, as per details provided in the note below.

id)

#Higher sample size should be used even for assays claiming 99% specificity.

1026 *In the absence of natural samples, spiked samples may be used, as per details provided in the note below. 1027 Note:
1028 Recombinant NS1 antigen of cross reactive flaviviruses (Zika, West Nile and Japanese Encephalitis viruses) expressed 1029 in mammalian cells can be obtained commercially and reconstituted in serum samples (100 ng -1 µg/ml) and diluted 1030 in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1). 1031 Before used for evaluation, flavivirus NS1 reconstituted in serum samples needs to be tested by the dengue NS1 1032 reference assay, and dilutions which are negative for dengue should be used for evaluation. 1033 The serum samples used for reconstitution should be negative for Dengue NS1, RNA and IgM antibody. 1034 6. Test reproducibility 1035 1036 1037 as follows:
1038 1039 1040 1041 1042 samples).
1043 1044 1045

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 47 | Page

1046 1047

1048

1049

1050 7. Acceptance Criteria
1051 Expected sensitivity: ≥90% 1052 Expected specificity: ≥95% 1053 Cross reactivity with other flavivirus antigens: Nil 1054 9. Publication Rights: 1055 1056

1057

1058 1059 1060 1061 1062

1063

ity: ≥90% 1052 Expected specificity: ≥95% 1053 Cross reactivity with other flavivirus antigens: Nil 1054 9. Publication Rights: 1055 1056

1057

1058 1059 1060 1061 1062

1063

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 48 | Page

VI. References: 1064

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian 1065 E, Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, 1066 Peeling RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection 1067 of Dengue virus NS1 antigen and anti-Dengue virus IgM antibody. PLoS Negl Trop Dis. 2014 Oct 1068 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 1069
2. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, 1070 Nisalak A, Yoon IK, Fernandez S. Evaluation of a Dengue NS1 antigen detection assay 1071 sensitivity and specificity for the diagnosis of acute Dengue virus infection. PLoS Negl 1072 Trop Dis. 2014 Oct 2;8(10):e3193. doi: 10.1371/journal.pntd.0003193. 1073
3. Central Drugs Standard Control Organization. In-Vitro Diagnostic (IVD) Medical Devices Frequently 1074 Asked Questions.

Available at: 1075 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/IVD/FAQs/CDSCO-IVD- 1076 FAQ-03-2022-.pdf
1077 4. U.S. Food and Drug Administration. Dengue Virus Serological Reagents - Class II Special Controls 1078 Guideline for Industry and Food and Drug Administration Staff. 2014.

AQ-03-2022-.pdf
1077 4. U.S. Food and Drug Administration. Dengue Virus Serological Reagents - Class II Special Controls 1078 Guideline for Industry and Food and Drug Administration Staff. 2014. Available at: 1079 https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation- 1080 emitting-products/Dengue-virus-serological-reagents-class-ii-special-controls-guideline-industry- 1081 and-food-and-drug
1082 5. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 1083 Diagnostic Assessment TGS-3. 2017. Available at: 1084 1085 eng.pdf;sequence=1
1086

1087 VII. 1088

1089

1090

1091

1092

1093

1094

1095

1096

1097

1098

1099

1100

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 49 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE NS1 ELISA KIT 1101 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: strong, moderate, weak)
Negative samples (provide details: clinical/spiked, including cross reactivity panel)

1102 Results 1103

Positive Negative Total Name of Dengue NS1 - based ELISA kit Positive

Negative

Total

1104

Estimate (%) 95% CI Sensitivity

Specificity

1105 ● Details of cross reactivity with other flavivirus NS1 antigens:
1106 ● Conclusions: 1107 o Sensitivity, specificity 1108 o Performance: Satisfactory / Not satisfactory 1109

ty

1105 ● Details of cross reactivity with other flavivirus NS1 antigens:
1106 ● Conclusions: 1107 o Sensitivity, specificity 1108 o Performance: Satisfactory / Not satisfactory 1109

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 50 | Page

(Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from the batch 1110 mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 1111 Disclaimers 1112 1113 1114 Note: This report is exclusively for …………. Kit (Lot No……) manufactured by …………… (Supplied 1115 by ……….) 1116 1117 1118 Signature of Director/ Director-In-charge …………………… Seal ……………………… 1119 ****End of the Report 1120

1121

1122

1123

1124

1125

1126

1127

1128

1129

1130

1131

1132

1133

1134

1135

1136

1137

1138

1139

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 51 | Page

Field evaluation protocol for Dengue NS1 ELISA kits 1140 I. Background:
1141 1142 1143 1144 1145 II. Purpose: 1146 To evaluate the performance characteristics of Dengue NS1 ELISA kits in the diagnosis of Dengue 1147 infection in individuals with unknown disease status. 1148 III. Requirements:
1149 1150 1151 1152 1153 1154 4. Laboratory supplies
1155

1156 IV. Ethical approval:
1157 The study will be initiated after approval from the institutional human ethics committee.
1158 V. Procedure: 1159 1.

1154 4. Laboratory supplies
1155

1156 IV. Ethical approval:
1157 The study will be initiated after approval from the institutional human ethics committee.
1158 V. Procedure: 1159

1. Study design/type: Cross-sectional study 1160 1161 1162 1163 1164 1165 1166 1167 1168 ⮚ Handling of Dengue NS1 ELISA kits received for performance evaluation 1169 1170 1171 1172

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 52 | Page

3. Sample size for performance evaluation: 1173 Sample sizes of positive and negative samples of Dengue against different values of 1174 sensitivity and specificity are provided in Tables 1 and 2. Sample sizes have been calculated 1175 assuming 95% level of significance, and an absolute precision of 5%. It is further assumed 1176 that 30% of the individuals attending the health care facilities for acute febrile illness and 1177 suspected for Dengue will be positive for Dengue. Appropriate sample size has to be chosen 1178 from the tables according to the values of sensitivity and specificity being claimed by the 1179 manufacturer. If a claimed sensitivity/specificity is not present in the table, the 1180 manufacturer needs to consider the sample size associated with the largest 1181 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as 1182 per the next smaller value of the sensitivity/ specificity available in the table). For example, 1183 if a manufacturer claims a sensitivity of 93%, they are required to use a sample size 1184 mentioned against 90% sensitivity.

e sensitivity/ specificity available in the table). For example, 1183 if a manufacturer claims a sensitivity of 93%, they are required to use a sample size 1184 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 1185 usage of the sample size outlined for 85% specificity.
1186 Sample size has to be calculated based on both the sensitivity and the specificity. The 1187 final sample size will be the maximum of the two. For example, at 95% sensitivity and 1188 95% specificity, the sample size required will be 245 (maximum of 245 and 105).
1189

1190 Table 1. Sample sizes for different values of sensitivity claimed by the manufacturer. 1191 Sensitivity Calculated sample size No. of individuals* off] 99%# 51 55 95% 243 245 90% 461 465 85% 653 655 80% 820 820

• Individuals attending the health care facilities for

1192 #Higher sample size should be used even for assays claiming 99% sensitivity. 1193

1194 Table 2. Sample sizes for different values of specificity claimed by the manufacturer. 1195 Specificity Calculated sample size No. of individuals* off] 99%# 22 25 95% 104 105 90% 198 200 85% 280 280 80% 351 355

nt values of specificity claimed by the manufacturer. 1195 Specificity Calculated sample size No. of individuals* off] 99%# 22 25 95% 104 105 90% 198 200 85% 280 280 80% 351 355

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 53 | Page

• Individuals attending the health care facilities for

1196 #Higher sample size should be used even for assays claiming 99% specificity. 1197 Recruitment of cases shall be halted once desired number of positive and negative samples are 1198 reached. 1199

1200 4. Inclusion criteria: 1201 Individuals with Dengue like illness (A patient with acute febrile illness of 2-7 days with two or 1202 more manifestations: Head ache, retro-orbital pain, myalgia, arthralgia, rash, hemorrhagic 1203 manifestations) 1204 5. Exclusion criteria 1205 Individuals with already known positive history for other pathogens 1206 6. Reference assay:
1207 US-FDA approved Dengue NS1 ELISA kit should be used as reference assay. 1208 Serotype status to be assessed using CDC / NIV real-time PCR serotyping protocols.
1209 7. Study implementation: 1210 The individuals with Dengue like illness will be recruited into the study and five ml of whole blood 1211 will be collected in vacutainer tubes and the serum will be separated by centrifugation and used 1212 for the study. The serum sample will be subjected to the following reference tests and the index 1213 test. 1214 It needs to be ensured that the samples are tested by reference tests and index test simultaneously. 1215 8.

ple will be subjected to the following reference tests and the index 1213 test. 1214 It needs to be ensured that the samples are tested by reference tests and index test simultaneously. 1215 8. Positive samples: 1216 Samples positive by the reference NS1 ELISA assay and real-time PCR assay (True positives). 1217 will be considered as true positive sample.
1218 9. Negative samples:
1219 Samples negative by the reference NS1 ELISA assay and real-time PCR using CDC/NIV 1220 serotyping protocol will be considered as true negative. 1221 A. Cross reactivity: 1222 Clinical samples or commercially available NS1 antigens from other flaviviruses will be used to 1223 test cross reactivity of the index test. 1224

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 54 | Page

1. Japanese Encephalitis PCR/antigen positive: 5 samples 1225
2. West Nile Virus PCR/antigen: 5 samples 1226
3. Zika Virus PCR/antigen: 5 samples 1227 *In the absence of natural samples, spiked samples may be used, as per details provided in the note below. 1228 Note:
   1229 Recombinant NS1 antigen of cross reactive flaviviruses (Zika, West Nile and Japanese Encephalitis viruses) expressed 1230 in mammalian cells can be obtained commercially and reconstituted in serum samples (100 ng -1 µg/ml) and diluted 1231 in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1). 1232 Before used for evaluation, NS1 reconstituted in serum samples needs to be tested by the reference assay and dilution 1233 which are positive only should be used for evaluation.

ecific NS1). 1232 Before used for evaluation, NS1 reconstituted in serum samples needs to be tested by the reference assay and dilution 1233 which are positive only should be used for evaluation. 1234 The serum samples used for reconstitution should be negative for Dengue NS1, RNA and IgM antibody. 1235 10. Statistical analysis:
1236 Sensitivity and specificity will be calculated. 1237 Interim analysis of data shall be conducted on completing evaluation of 25%, 50% and 75% of 1238 samples. If, at any point, the performance of the assay is found to be not satisfactory, the assay 1239 shall not be evaluated further. Evaluation fee shall be charged accordingly. 1240

1241 11. Test reproducibility 1242 a. Sample size for lot-to-lot reproducibility 1243 1244 as follows:
1245 1246 1247 1248 1249 samples).
1250 1251 1252

1253 1254

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 55 | Page

1255

1256

1257 12. Acceptance Criteria 1258 Expected sensitivity: ≥90% 1259 Expected specificity: ≥95% 1260 Cross-reactivity with other flavivirus antigens: Nil 1261 13. Publication Rights: 1262 1263

1264 1265 1266 1267 1268

1269 VI. References: 1270

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian 1271 E, Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, 1272 Peeling RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection 1273

Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, 1272 Peeling RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection 1273

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 56 | Page

of Dengue virus NS1 antigen and anti-Dengue virus IgM antibody. PLoSNegl Trop Dis. 2014 Oct 1274 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 1275 2. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, 1276 Nisalak A, Yoon IK, Fernandez S. Evaluation of a Dengue NS1 antigen detection assay 1277 sensitivity and specificity for the diagnosis of acute Dengue virus infection. PLoSNegl 1278 Trop Dis. 2014 Oct 2;8(10):e3193. doi: 10.1371/journal.pntd.0003193. 1279 3. Ganeshkumar P, Murhekar MV, Poornima V, Saravanakumar V, Sukumaran K, 1280 Anandaselvasankar A, John D, Mehendale SM. Dengue infection in India: A systematic 1281 review and meta-analysis. PLoSNegl Trop Dis. 2018 Jul 16;12(7):e0006618. doi: 1282 10.1371/journal.pntd.0006618. 1283

1284 4. Castro-Jorge LA, Machado PR, Fávero CA, Borges MC, Passos LM, de Oliveira RM, 1285 Fonseca BA. Clinical evaluation of the NS1 antigen-capture ELISA for early diagnosis of 1286 Dengue virus infection in Brazil. J Med Virol. 2010 Aug;82(8):1400-5. doi: 1287 10.1002/jmv.21814. 1288

1289 5. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 1290 Diagnostic Assessment TGS-3. 2017. Available at: 1291 1292 eng.pdf;sequence=1
1293

1294 VII.

orld Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 1290 Diagnostic Assessment TGS-3. 2017. Available at: 1291 1292 eng.pdf;sequence=1
1293

1294 VII. 1295

1296

1297

1298

1299

1300

1301

1302

1303

1304

1305

1306

1307

1308

1309

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 57 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE NS1 ELISA KIT 1310 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples: Not applicable, may categorize cases as per duration of illness Negative samples (may categorize as per duration of illness, must include cross reactivity panel)

1311 Results 1312

Positive Negative Total Name of Dengue NS1 based ELISA kit Positive

Negative

Total

1313

Estimate (%) 95% CI Sensitivity

Specificity

1314 ● Details of cross reactivity with other flavivirus NS1 antigens:
1315 ● Conclusions: 1316 o Sensitivity, specificity 1317 o 1318

te (%) 95% CI Sensitivity

Specificity

1314 ● Details of cross reactivity with other flavivirus NS1 antigens:
1315 ● Conclusions: 1316 o Sensitivity, specificity 1317 o 1318

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 58 | Page

(Sensitivity and specificity have been assessed in using kits provided by the manufacturer from the batch mentioned above using 1319 ….. sample in controlled lab setting. Results should not be extrapolated to other sample types.) 1320

1321 Disclaimers 1322 1323 1324 Note: This report is exclusively for NS1…………………Kit (Lot No……) manufactured by …………… (supplied 1325 by ……….) 1326 1327 1328 Signature of Director/ Director-In charge …………………… Seal …………………………….. 1329 ****End of the Report 1330

1331

1332

1333

1334

1335

1336

1337

1338

1339

1340

1341

1342

1343

1344

1345

1346

1347

1348

1349

f the Report**************************** 1330

1331

1332

1333

1334

1335

1336

1337

1338

1339

1340

1341

1342

1343

1344

1345

1346

1347

1348

1349

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 59 | Page

Performance evaluation protocol for Dengue IgM RDT kits 1350 I. Background:
1351 1352 1353 1354 1355 II. Purpose: 1356 To evaluate the performance characteristics of Dengue IgM RDT kits in the diagnosis of Dengue 1357 infection. 1358 III. Requirements:
1359 a) Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 1360 1361 1362 b) Evaluation sites/laboratories (With required equipment) 1363 c) Reference test kits 1364 d) Characterised Evaluation panel 1365 e) Laboratory supplies
1366 IV. Ethical approvals:
1367 1368 1369 1370 V. Procedure: 1371

1. Study design/type: Diagnostic accuracy study using archived/ leftover clinical samples 1372 1373 1374 1375 1376 1377 1378 1379 1380 ⮚ Handling of Dengue IgM Rapid IVD kits received for performance evaluation 1381 1382

ccuracy study using archived/ leftover clinical samples 1372 1373 1374 1375 1376 1377 1378 1379 1380 ⮚ Handling of Dengue IgM Rapid IVD kits received for performance evaluation 1381 1382

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 60 | Page

1383 1384

1385 3. Preparation of Dengue IgM Rapid IVD kit evaluation panel 1386 Well characterised Dengue IVD kit evaluation panel is a critical requirement for performance 1387 evaluation of IVD kits. Hence statistically significant number of sera samples should be 1388 available from Dengue confirmed cases. Further characterised for Dengue IgM positivity by 1389 using approved reference kits having high sensitivity and specificity. 1390 Dengue IgM performance evaluation panel need to be tested again by the reference assays at 1391 the time of evaluating a particular index test to confirm the positive and negative status of the 1392 samples. 1393 4. Reference assay:
1394 US-FDA approved Dengue IgM ELISA kit should be used as reference assay. 1395 NS1 antigen status to be assessed using US FDA approved NS1 ELISA kit. 1396 Serotype status to be assessed using a combination of CDC/NIV real-time PCR serotyping 1397 protocols.
1398 At least 50% of the samples should be positive by real-time PCR or NS1 antigen and IgM 1399 ELISA. 1400 Primary and Secondary status to be assessed by Panbio Dengue IgG capture ELISA kit. 1401

1402 5.

0% of the samples should be positive by real-time PCR or NS1 antigen and IgM 1399 ELISA. 1400 Primary and Secondary status to be assessed by Panbio Dengue IgG capture ELISA kit. 1401

1402 5. Sample size and sample panel composition: Sample sizes of positive and negative 1403 samples of Dengue against different values of sensitivity and specificity are provided in Tables 1404 1 and 2. Sample sizes have been calculated assuming 95% level of significance, an absolute 1405 precision of 5%, and invalid test rate ≤5%. Appropriate sample size has to be chosen from the 1406 tables according to the values of sensitivity and specificity being claimed by the manufacturer. 1407 If a claimed sensitivity/specificity is not present in the table, the manufacturer needs to consider 1408 the sample size associated with the largest sensitivity/specificity provided in the table that is 1409 smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ specificity 1410 available in the table). For example, if a manufacturer claims a sensitivity of 93%, they are 1411 required to use a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% 1412 specificity would require usage of the sample size outlined for 85% specificity.
1413

1414 Positive samples: The panel of positive samples should include samples positive by the reference 1415 assay, with 50% sampls positive for Dengue NS1/RT-PCR assay (True positives). Samples should 1416 be representative of all 4 serotypes and varying degrees of positivity.

ive by the reference 1415 assay, with 50% sampls positive for Dengue NS1/RT-PCR assay (True positives). Samples should 1416 be representative of all 4 serotypes and varying degrees of positivity. The samples should be 1417 classified as strong, moderate and weak positives based on ELISA units of the reference assay. 1418

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 61 | Page

1419 Negative samples: These should include samples negative by the reference assay, NS1 ELISA 1420 assay and/or real-time PCR using CDC/NIV serotyping protocol (True negatives). 1421

1422 Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 1423 sensitivity claimed by the manufacturer. 1424 Sensitivity Calculated sample size No. of Positive Samples required off] 99%# 16 20 Strong Positive: 6 Moderate Positive: 8 Weak Positive: 6 95% 77 80 Strong Positive: 23 Moderate Positive: 34 Weak Positive: 23 90% 145 150 Strong Positive: 43 Moderate Positive: 64 Weak Positive: 43 85% 206 210 Strong Positive: 61 Moderate Positive: 88 Weak Positive: 61 80% 258 260 Strong Positive: 75 Moderate Positive: 110 Weak Positive: 75

1425 #Higher sample size should be used even for assays claiming 99% sensitivity. 1426

1427 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 1428 specificity claimed by the manufacturer. 1429 Specificity Calculated sample size No.

1427 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 1428 specificity claimed by the manufacturer. 1429 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off] 99%# 16 20 Chikungunya positive: 4
aAcute febrile cases: 5
*Japanese Encephalitis IgM positive: 1
*West Nile Virus IgM positive: 1 *Zika Virus IgM positive: 1

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 62 | Page

Rheumatoid Arthritis/other autoimmune disease cases: 4 bHealthy subjects from endemic regions: 4
95% 77 80 Chikungunya positive: 16
aAcute febrile cases: 23
*Japanese Encephalitis IgM positive: 3
*West Nile Virus IgM positive: 3 *Zika Virus IgM positive: 3 disease cases: 16 bHealthy subjects from endemic regions: 16
90% 145 150 Chikungunya positive: 30
aAcute febrile cases: 45
*Japanese Encephalitis IgM positive: 5
*West Nile Virus IgM positive: 5 *Zika Virus IgM positive: 5 disease cases: 30
bHealthy subjects from endemic regions: 30
85% 206 210 Chikungunya positive: 42
aAcute febrile cases: 63
*Japanese Encephalitis IgM positive: 7
*West Nile Virus IgM positive: 7 *Zika Virus IgM positive: 7 disease cases: 42 bHealthy subjects from endemic regions: 42
80% 258 260 Chikungunya positive: 52
aAcute febrile cases: 77
*Japanese Encephalitis IgM positive: 9
*West Nile Virus IgM positive: 9 *Zika Virus IgM positive: 9 disease cases: 52 bHealthy subjects from endemic regions: 52
a Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR)

9
*West Nile Virus IgM positive: 9 *Zika Virus IgM positive: 9 disease cases: 52 bHealthy subjects from endemic regions: 52
a Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, RNA)

1430 #Higher sample size should be used even for assays claiming 99% specificity. 1431 *Note: Depending on the availability of IgM positive samples for cross reactive flaviviruses, the requirement of 1432 samples for each virus may be increased or decreased accordingly to reach the total number of samples. If IgM positive 1433 samples for cross reactive flaviviruses are not available, commercially available IgM sera panel for different viruses 1434 can be procured and used to test cross reactivity. 1435 6. Test reproducibility 1436

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 63 | Page

1437 1438 as follows:
1439 1440 1441 1442 1443 samples).
1444 1445 1446

1447 1448

1449

1450

1451 B. Sample size for reader-to-reader reproducibility 1452 For reader-to-reader reproducibility, sample size should be 25 (15 positive samples comprising 10 1453 low positive AND 5 moderate/high positive samples, and 10 negative samples). 1454

1455 Two operators will be reading the test results independently as per manufacturer’s instruction. 1456 Agreement should be 100% between the operators. 1457 7. Acceptance Criteria
1458

4

1455 Two operators will be reading the test results independently as per manufacturer’s instruction. 1456 Agreement should be 100% between the operators. 1457 7. Acceptance Criteria
1458

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 64 | Page

Expected sensitivity: ≥80% 1459 Expected specificity: ≥90% 1460 1461 8. Publication Rights: 1462 1463

1464 1465 1466 1467 1468 VI. References: 1469

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Pelegrino JL, Vázquez S, 1470 Artsob H, Drebot M, Gubler DJ, Halstead SB, Guzmán MG, Margolis HS, Nathanson CM, Rizzo Lic 1471 NR, Bessoff KE, Kliks S, Peeling RW. Evaluation of commercially available anti-Dengue virus 1472 immunoglobulin M tests. Emerg Infect Dis. 2009 Mar;15(3):436-40. doi: 10.3201/eid1503.080923.
   1473
2. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian E, 1474 Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, Peeling 1475 RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection of 1476 Dengue virus NS1 antigen and anti-Dengue virus IgM antibody. PLoS Negl Trop Dis. 2014 Oct 1477 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 1478
3. WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. 1479 (Diagnostics evaluation series, 3). ISBN 978 92 4 159775 3.
   1480
4. Central Drugs Standard Control Organization. Guidance on Performance Evaluation of In-vitro 1481 Diagnostic Medical Devices.

tics evaluation series, 3). ISBN 978 92 4 159775 3.
1480 4. Central Drugs Standard Control Organization. Guidance on Performance Evaluation of In-vitro 1481 Diagnostic Medical Devices. 2018. Available at: 1482 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/medical 1483 device/guidanceperformanceivd.pdf 1484 5. Central Drugs Standard Control Organization. In-Vitro Diagnostic (IVD) Medical Devices Frequently 1485 Asked Questions. 2022. Available at: 1486 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/IVD/FAQs/CDSCO-IVD- 1487 FAQ-03-2022-.pdf
1488 6. U.S. Food and Drug Administration. Dengue Virus Serological Reagents - Class II Special Controls 1489 Guideline for Industry and Food and Drug Administration Staff. 2014. Available at: 1490 https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation- 1491 emitting-products/Dengue-virus-serological-reagents-class-ii-special-controls-guideline-industry- 1492 and-food-and-drug
1493 7. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 1494 Diagnostic Assessment TGS-3. 2017. Available at: 1495 1496 eng.pdf;sequence=1
1497

1493 7. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 1494 Diagnostic Assessment TGS-3. 2017. Available at: 1495 1496 eng.pdf;sequence=1
1497

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 65 | Page

8. Yow KS, Aik J, Tan EY, Ng LC, Lai YL. Rapid diagnostic tests for the detection of recent Dengue 1498 infections: An evaluation of six kits on clinical specimens. PLoS One. 2021 Apr 1;16(4):e0249602. 1499 doi: 10.1371/journal.pone.0249602. 1500

1501 *The validation protocols need to be revisited after introduction of Dengue vaccines and the 1502 acceptance criteria needs revisiting every year so as to enable the availability of best 1503 diagnostic kits.
1504 VII. 1505

1506

1507

1508

1509

1510

1511

1512

1513

1514

1515

1516

1517

1518

1519

1520

1521

1522

1523

1524

1525

1526

1527

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 66 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE IgM RDT KIT 1528 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No.

age

PERFORMANCE EVALUATION REPORT FOR DENGUE IgM RDT KIT 1528 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: strong, moderate, weak)
Negative samples (provide details: clinical/spiked, including cross reactivity panel)

1529 Results:
1530

Positive Negative Total Name of Dengue antibody - based RDT kit Positive

Negative

Total

1531

Estimate (%) 95% CI Sensitivity

Specificity

Conclusions: 1532 o Sensitivity, specificity 1533 o Performance: Satisfactory / Not satisfactory 1534 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 1535 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 1536 Disclaimers 1537

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 67 | Page

1538 1539 Note: This report is exclusively for ………………………Kit (Lot No……) manufactured by …………… 1540 (Supplied by ……….) 1541 1542 1543 Signature of Director/ Director-In-charge …………………… Seal ………………………. 1544

1545 ****End of the Report 1546

1547

1548

1549

1550

1551

1552

1553

1554

1555

1556

1557

1558

1559

1560

1561

1562

1563

1564

1565

1566

port**************************** 1546

1547

1548

1549

1550

1551

1552

1553

1554

1555

1556

1557

1558

1559

1560

1561

1562

1563

1564

1565

1566

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 68 | Page

Performance evaluation protocol for Dengue IgM ELISA kits 1567 I. Background:
1568 1569 1570 1571 1572 II. Purpose: 1573 To evaluate the performance characteristics of Dengue IgM ELISA kits in the diagnosis of Dengue 1574 infection. 1575 III. Requirements:
1576 1577 1578 1579 1580 1581 1582 1583 IV. Ethical approval:
1584 1585 1586 1587 V. Procedure: 1588

1. Study design/type: Diagnostic accuracy study using archived/leftover clinical samples. 1589 1590 1591 1592 1593 1594 1595 1596 1597 ⮚ Handling of Dengue IgM ELISA IVD kits received for performance evaluation 1598 1599

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 69 | Page

1600 1601 3. Preparation of Dengue IgM ELISA IVD kit evaluation panel 1602 Well characterised Dengue IVD kit evaluation panel is a critical requirement for performance 1603 evaluation of IVD kits. Hence statistically significant number of sera samples should be 1604 available from Dengue confirmed cases. Further characterised for Dengue IgM positivity by 1605 using approved reference kits having high sensitivity and specificity. 1606 Dengue IgM performance evaluation panel need to be tested again by the reference assays at 1607 the time of evaluating a particular index test to confirm the positive and negative status of the 1608 samples.

erformance evaluation panel need to be tested again by the reference assays at 1607 the time of evaluating a particular index test to confirm the positive and negative status of the 1608 samples. 1609 4. Reference assay:
1610 US-FDA approved Dengue IgM ELISA kit should be used as reference assay. 1611 NS1 antigen status to be assessed using US FDA approved NS1 ELISA kit. 1612 Serotype status to be assessed using a combination of CDC/NIV real-time PCR serotyping 1613 protocols.
1614 At least 50% of the samples should be positive by real-time PCR or NS1 antigen and IgM 1615 ELISA. 1616 Primary and Secondary status to be assessed by Panbio Dengue IgG capture ELISA kit. 1617 5. Sample size and sample panel composition: Sample sizes of positive and negative 1618 samples and sample panel composition against different values of sensitivity and specificity are 1619 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of 1620 significance, and an absolute precision of 5%. Appropriate sample size has to be chosen from 1621 the tables according to the values of sensitivity and specificity being claimed by the 1622 manufacturer. If a claimed sensitivity/specificity is not present in the table, the manufacturer 1623 needs to consider the sample size associated with the largest sensitivity/specificity provided in 1624 the table that is smaller to the claimed value (that is, as per the next smaller value of the 1625 sensitivity/ specificity available in the table).

rgest sensitivity/specificity provided in 1624 the table that is smaller to the claimed value (that is, as per the next smaller value of the 1625 sensitivity/ specificity available in the table). For example, if a manufacturer claims a sensitivity 1626 of 93%, they are required to use a sample size mentioned against 90% sensitivity. Similarly, a 1627 claim of 87% specificity would require usage of the sample size outlined for 85% specificity.
1628

1629 Positive samples: The panel of positive samples should include samples positive by the reference 1630 assay, with 50% samples positive for Dengue NS1/ RT-PCR assay (True positives). Samples 1631 should be representative of primary/secondary Dengue and all 4 Dengue virus serotypes, with 1632 varying degrees of positivity. The samples should be classified as strong, moderate and weak 1633 positives based on ELISA units of the reference assay. 1634

1635

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 70 | Page

Negative samples: These should include samples negative by the reference assay, NS1 ELISA 1636 and/or real-time PCR using CDC and/or NIV serotyping protocols. (True negatives). 1637

1638 Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 1639 sensitivity claimed by the manufacturer. 1640 Sensitivity Calculated sample size No.

1638 Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 1639 sensitivity claimed by the manufacturer. 1640 Sensitivity Calculated sample size No. of Positive Samples required off] 99%# 15 20 Strong Positive: 4 Moderate Positive: 8 Weak Positive: 8 95% 73 80 Strong Positive: 18 Moderate Positive: 31 Weak Positive: 31 90% 138 140 Strong Positive: 30 Moderate Positive: 55 Weak Positive: 55 85% 196 200 Strong Positive: 42 Moderate Positive: 79 Weak Positive: 79 80% 246 250 Strong Positive: 54 Moderate Positive: 98 Weak Positive: 98

1641 #Higher sample size should be used even for assays claiming 99% sensitivity. 1642 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 1643 specificity claimed by the manufacturer. 1644 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off] 99%# 15 20 Chikungunya positive: 3
aAcute febrile cases: 6
*Japanese Encephalitis IgM positive: 1
*West Nile Virus IgM positive: 1
*Zika Virus IgM positive: 1

mple size rounded off] 99%# 15 20 Chikungunya positive: 3
aAcute febrile cases: 6
*Japanese Encephalitis IgM positive: 1
*West Nile Virus IgM positive: 1
*Zika Virus IgM positive: 1

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 71 | Page

disease cases: 4
bHealthy subjects from endemic regions: 4
95% 73 80 Chikungunya positive: 10
aAcute febrile cases: 25
*Japanese Encephalitis IgM positive: 5
*West Nile Virus IgM positive: 5
*Zika Virus IgM positive: 5
disease cases: 15
bHealthy subjects from endemic regions: 15
90% 138 140 Chikungunya positive: 18
aAcute febrile cases: 43
*Japanese Encephalitis IgM positive: 9
*West Nile Virus IgM positive: 9
*Zika Virus IgM positive: 9
disease cases: 26
bHealthy subjects from endemic regions: 26
85% 196 200 Chikungunya positive: 25
aAcute febrile cases: 63
*Japanese Encephalitis IgM positive: 12
*West Nile Virus IgM positive: 12
*Zika Virus IgM positive: 12
disease cases: 38
bHealthy subjects from endemic regions: 38
80% 246 250 Chikungunya positive: 31
aAcute febrile cases: 77
*Japanese Encephalitis IgM positive: 16 *West Nile Virus IgM positive: 16
*Zika Virus IgM positive: 16
disease cases: 47
bHealthy subjects from endemic regions: 47
a Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, RNA)

1645 #Higher sample size should be used even for assays claiming 99% specificity.

PCR) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, RNA)

1645 #Higher sample size should be used even for assays claiming 99% specificity. 1646 *Note: Depending on the availability of IgM positive samples for cross reactive flaviviruses, the requirement of 1647 samples for each virus may be increased or decreased accordingly to reach the total number of samples. If IgM positive 1648 samples for cross reactive flaviviruses are not available, commercially available IgM sera panel for different viruses 1649 can be procured and used to test cross reactivity. 1650 6. Test reproducibility 1651

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 72 | Page

1652 1653 as follows:
1654 1655 1656 1657 1658 samples).
1659 1660 1661

1662 1663

1664

1665

1666 7. Acceptance criteria 1667 Expected sensitivity: ≥90% 1668 Expected specificity: ≥95%
1669 8. Publication Rights: 1670 1671

.
1659 1660 1661

1662 1663

1664

1665

1666 7. Acceptance criteria 1667 Expected sensitivity: ≥90% 1668 Expected specificity: ≥95%
1669 8. Publication Rights: 1670 1671

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 73 | Page

1672

1673 1674 1675 1676 1677 VI. References: 1678

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Pelegrino JL, 1679 Vázquez S, Artsob H, Drebot M, Gubler DJ, Halstead SB, Guzmán MG, Margolis HS, 1680 Nathanson CM, Rizzo Lic NR, Bessoff KE, Kliks S, Peeling RW. Evaluation of 1681 commercially available anti-Dengue virus immunoglobulin M tests. Emerg Infect Dis. 1682 2009 Mar;15(3):436-40. doi: 10.3201/eid1503.080923.
   1683
2. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian 1684 E, Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, 1685 Peeling RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection 1686 of Dengue virus NS1 antigen and anti-Dengue virus IgM antibody. PLoSNegl Trop Dis. 2014 Oct 1687 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 1688
3. WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. 1689 (Diagnostics evaluation series, 3). ISBN 978 92 4 159775 3.
   1690
4. Central Drugs Standard Control Organization. Guidance on Performance Evaluation of In-vitro 1691 Diagnostic Medical Devices.

tics evaluation series, 3). ISBN 978 92 4 159775 3.
1690 4. Central Drugs Standard Control Organization. Guidance on Performance Evaluation of In-vitro 1691 Diagnostic Medical Devices. 2018. Available at: 1692 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/medical 1693 device/guidanceperformanceivd.pdf 1694 5. Central Drugs Standard Control Organization. In-Vitro Diagnostic (IVD) Medical Devices Frequently 1695 Asked Questions. 2022. Available at: 1696 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/IVD/FAQs/CDSCO-IVD- 1697 FAQ-03-2022-.pdf
1698 6. U.S. Food and Drug Administration. Dengue Virus Serological Reagents - Class II Special Controls 1699 Guideline for Industry and Food and Drug Administration Staff. 2014. Available at: 1700 https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation- 1701 emitting-products/Dengue-virus-serological-reagents-class-ii-special-controls-guideline-industry- 1702 and-food-and-drug
1703 7. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 1704 Diagnostic Assessment TGS-3. 2017. Available at: 1705 1706 eng.pdf;sequence=1
1707

1708 *The validation protocols need to be revisited after introduction of Dengue vaccines and the 1709 acceptance criteria needs revisiting every year so as to enable the availability of best 1710 diagnostic kits.
1711 VII. 1712

be revisited after introduction of Dengue vaccines and the 1709 acceptance criteria needs revisiting every year so as to enable the availability of best 1710 diagnostic kits.
1711 VII. 1712

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 74 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE IgM ELISA KIT 1713 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: strong, moderate, weak)
Negative samples (provide details: clinical/spiked, including cross reactivity panel)

1714 Results:
1715

Positive Negative Total Name of Dengue antibody -based ELISA kit Positive

Negative

Total

1716

Estimate (%) 95% CI Sensitivity

Specificity

Conclusions: 1717 o Sensitivity, specificity 1718 o 1719 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from the batch 1720 mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 1721 Disclaimers 1722 1723 1724

etting using kits provided by the manufacturer from the batch 1720 mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 1721 Disclaimers 1722 1723 1724

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 75 | Page

Note: This report is exclusively for ………………………Kit (Lot No……) manufactured by …………… (Supplied 1725 by ……….) 1726 1727 1728 Signature of Director/ Director-In-charge …………………… Seal……………………………………… 1729

1730 ****End of the Report 1731

1732

1733

1734

1735

1736

1737

1738

1739

1740

1741

1742

1743

1744

1745

1746

1747

1748

1749

1750

1751

1752

1753

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 76 | Page

Performance evaluation protocol for Dengue NS1/IgM combo RDT kits 1754 I. Background:
1755 1756 1757 1758 1759 II. Purpose: 1760 To evaluate the performance characteristics of Dengue NS1/IgM combo RDT kits in the diagnosis 1761 of Dengue infection. 1762 III. Requirements:
1763 1764 1765 1766 1767 1768 1769 1770 IV. Ethical approvals:
1771 1772 1773 1774 V. Procedure: 1775

1. Study design/type: Diagnostic accuracy study using archived/leftover clinical samples. 1776 1777 1778 1779 1780 1781 1782 1783 1784 ⮚ Handling of Dengue NS1/IgM combo IVD kits received for performance evaluation 1785 1786

acy study using archived/leftover clinical samples. 1776 1777 1778 1779 1780 1781 1782 1783 1784 ⮚ Handling of Dengue NS1/IgM combo IVD kits received for performance evaluation 1785 1786

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 77 | Page

1787 1788 3. Preparation of Dengue RDT IVD kit evaluation panel 1789 Well characterised Dengue RDT IVD kit evaluation panel is a critical requirement for performance 1790 evaluation of IVD kits. Hence statistically significant number of sera samples should be available 1791 from Dengue confirmed cases. Further characterised for Dengue NS1 and IgM positivity by using 1792 approved reference kits having high sensitivity and specificity. 1793 Dengue NS1/IgM performance evaluation panel need to be tested again by the reference assays at 1794 the time of evaluating a particular index test to confirm the positive and negative status of the 1795 samples. 1796 4. Reference assay:
1797 Anti-DENV IgM detection ELISA US-FDA approved kit 1798 AND/OR 1799 DENV NS1 ELISA US-FDA approved kit 1800 Serotype status to be assessed using a combination of CDC and/or NIV real-time PCR serotyping 1801 protocols.
1802 All positive samples need confirmation reference NS1/IgM ELISA assay and real-time PCR assay. 1803 Sample size and sample panel composition: Sample sizes of positive and negative samples of 1804 Dengue against different values of sensitivity and specificity are provided in Tables 1 and 2.

1803 Sample size and sample panel composition: Sample sizes of positive and negative samples of 1804 Dengue against different values of sensitivity and specificity are provided in Tables 1 and 2. 1805 Sample sizes have been calculated assuming 95% level of significance, an absolute precision of 1806 5%, and invalid test rate ≤5%. Appropriate sample size has to be chosen from the tables according 1807 to the values of sensitivity and specificity being claimed by the manufacturer. If a claimed 1808 sensitivity/specificity is not present in the table, the manufacturer needs to consider the sample 1809 size associated with the largest sensitivity/specificity provided in the table that is smaller to the 1810 claimed value (that is, as per the next smaller value of the sensitivity/ specificity available in the 1811 table). For example, if a manufacturer claims a sensitivity of 93%, they are required to use a sample 1812 size mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require usage 1813 of the sample size outlined for 85% specificity.
1814 Positive samples: Samples which are positive for IgM or NS1 or both by the reference assays will 1815 be considered as true positive samples. There should be representation of samples positive for all 1816 four serotypes.
1817

1818 Negative samples: These should include samples negative by all the reference assays and real-time 1819 PCR using CDC and/or NIV serotyping protocol (True negatives). 1820

1821

1817

1818 Negative samples: These should include samples negative by all the reference assays and real-time 1819 PCR using CDC and/or NIV serotyping protocol (True negatives). 1820

1821

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 78 | Page

Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 1822 sensitivity claimed by the manufacturer. 1823 Sensitivity Calculated sample size No. of Positive Samples required off for balanced allocation] 99%# 16 28 *NS1 positive and IgM negative: 8 *NS1 and IgM positive: 12 *NS1 negative and IgM positive: 8 95% 77 84 *NS1 positive and IgM negative: 24 *NS1 and IgM positive: 36 *NS1 negative and IgM positive: 24 90% 145 160 *NS1 positive and IgM negative: 44 *NS1 and IgM positive: 72 *NS1 negative and IgM positive: 44 85% 206 220 *NS1 positive and IgM negative: 60 *NS1 and IgM positive: 100 *NS1 negative and IgM positive: 60 80% 258 260 *NS1 positive and IgM negative: 72 *NS1 and IgM positive: 116 *NS1 negative and IgM positive: 72

• all 4 serotypes shall be represented
  Note:
  In the absence of natural samples, spiked samples may be used as per details provided below:

Recombinant NS1 antigen of cross reactive flaviviruses (Zika, West Nile and Japanese Encephalitis viruses) expressed in mammalian cells can be obtained commercially and reconstituted in serum samples (100 ng - 1 µg/ml) and diluted in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1).

n cells can be obtained commercially and reconstituted in serum samples (100 ng - 1 µg/ml) and diluted in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1). Before used for evaluation, flavivirus NS1 reconstituted in serum samples needs to be tested by the dengue NS1 reference assay, and dilutions which are negative for dengue should be used for evaluation. The serum samples used for reconstitution should be negative for Dengue NS1, RNA and IgM antibody.

#Higher sample size should be used even for assays claiming 99% sensitivity.

1824 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 1825 specificity claimed by the manufacturer. 1826

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 79 | Page

Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off for balanced allocation] 99%# 16 28 Chikungunya positive: 1
aAcute febrile cases: 11
*Japanese Encephalitis IgM positive: 1
*West Nile Virus IgM positive: 1 *Zika Virus IgM positive: 1 **Japanese Encephalitis NS1 positive: 1
**West Nile Virus NS1 positive: 1 **Zika Virus NS1 positive: 1 disease cases: 5 bHealthy subjects from endemic regions: 5
95% 77 84 Chikungunya positive: 3
aAcute febrile cases: 33
*Japanese Encephalitis IgM positive: 3
*West Nile Virus IgM positive: 3 *Zika Virus IgM positive: 3 **Japanese Encephalitis NS1 positive: 3
**West Nile Virus NS1 positive: 3 **Zika Virus NS1 positive: 3 disease cases: 15

itive: 3
*West Nile Virus IgM positive: 3 *Zika Virus IgM positive: 3 **Japanese Encephalitis NS1 positive: 3
**West Nile Virus NS1 positive: 3 **Zika Virus NS1 positive: 3 disease cases: 15 bHealthy subjects from endemic regions: 15
90% 145 160 Chikungunya positive: 5
aAcute febrile cases: 65
*Japanese Encephalitis IgM positive: 5
*West Nile Virus IgM positive: 5 *Zika Virus IgM positive: 5 **Japanese Encephalitis NS1 positive: 5
**West Nile Virus NS1 positive: 5 **Zika Virus NS1 positive: 5 disease cases: 30 bHealthy subjects from endemic regions: 30
85% 206 220 Chikungunya positive: 7
aAcute febrile cases: 89
*Japanese Encephalitis IgM positive: 7
*West Nile Virus IgM positive: 7 *Zika Virus IgM positive: 7

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 80 | Page

**Japanese Encephalitis NS1 positive: 7
**West Nile Virus NS1 positive: 7 **Zika Virus NS1 positive: 7 disease cases: 41 bHealthy subjects from endemic regions: 41
80% 258 260 Chikungunya positive: 8
aAcute febrile cases: 106
*Japanese Encephalitis IgM positive: 8
*West Nile Virus IgM positive: 8 *Zika Virus IgM positive: 8 **Japanese Encephalitis NS1 positive: 8
**West Nile Virus NS1 positive: 8 **Zika Virus NS1 positive: 8 disease cases: 49 bHealthy subjects from endemic regions: 49
a Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, RNA)

endemic regions: 49
a Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, RNA)

*Depending on the availability of IgM positive samples for cross reactive flaviviruses, the requirement of samples for each virus may be increased or decreased accordingly to reach the total number of samples. If IgM positive samples for cross reactive flaviviruses are not available, commercially available IgM sera panel for different viruses can be procured and used to test cross reactivity.

**Before used for evaluation, the NS1 reconstituted in serum samples needs to be tested by the reference assay and dilution which are positive only should be used for evaluation. The serum sample used for spiking or reconstitution should be negative for Dengue NS1, RNA and IgM antibody.
#Higher sample size should be used even for assays claiming 99% specificity.

1827 5. Test reproducibility 1828 1829 1830 as follows:
1831 1832 1833 1834 comprising 10 low positive AND 5 moderate/high positive samples with adequate 1835 representation of NS1 and IgM, and 10 negative samples).
1836 1837 1838

1839

s:
1831 1832 1833 1834 comprising 10 low positive AND 5 moderate/high positive samples with adequate 1835 representation of NS1 and IgM, and 10 negative samples).
1836 1837 1838

1839

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 81 | Page

1840

1841

1842

1843 B. Sample size for reader-to-reader reproducibility 1844 For reader-to-reader reproducibility, sample size should be 25 (15 positive samples comprising 10 1845 low positive AND 5 moderate/high positive samples, and 10 negative samples). 1846

1847 Two operators will be reading the test results independently as per manufacturer’s instruction. 1848 Agreement should be 100% between the operators. 1849 C. Interpretation of results 1850 Since the kits have been provided in combo format, concordance has to be calculated separately 1851 for NS1 and IgM, and the overall sensitivity and specificity have to be calculated based on the 1852 combined results of NS1 and IgM. If the sample is positive for any one or both analytes (NS1 or 1853 IgM or both), then the sample is considered positive. Refer the table below for interpretation: 1854 NS1 Reference test result IgM reference test result Final Reference test result NS1 Index test result IgM Index test result Final index test result Interpretation + + Positive +

Positive True Positive + + Positive

Positive True Positive + + Positive

Negative False Negative + + Positive + + Positive True Positive

pretation + + Positive +

Positive True Positive + + Positive

Positive True Positive + + Positive

Negative False Negative + + Positive + + Positive True Positive

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 82 | Page

Positive +

Positive True Positive +

Positive

Positive True Positive +

Positive

Negative False Negative

Positive +

Positive True Positive

Positive

Positive True Positive

Positive

Negative False Negative

Negative

Positive False Positive

Negative +

Positive False Positive

1855 6. Acceptance criteria: 1856 A minimum concordance of 80% for NS1 and 80% for IgM should be achieved with the reference 1857 assay, and an overall combined sensitivity* and specificity$ of ≥90% each. 1858 Cross reactivity with other flavivirus antigens: Nil 1859 1860

• Samples which are positive for NS1 or IgM or both by the kit under evaluation (irrespective of the 1861 reference assay results) will be considered as positive and used for sensitivity calculation 1862 $ Sample which are negative for both NS1 and IgM by kit under evaluation (irrespective of the reference 1863 assay results) will be considered as negative and used for specificity calculation 1864 9. Publication Rights: 1865 1866

1867 1868 1869 1870 1871

1872 VI. References: 1873 1.

3 assay results) will be considered as negative and used for specificity calculation 1864 9. Publication Rights: 1865 1866

1867 1868 1869 1870 1871

1872 VI. References: 1873

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian E, Pelegrino 1874 JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, Peeling RW, Margolis HS. 1875 Evaluation of commercially available diagnostic tests for the detection of Dengue virus NS1 antigen and anti- 1876 Dengue virus IgM antibody. PLoSNegl Trop Dis. 2014 Oct 16;8(10):e3171. doi: 1877 10.1371/journal.pntd.0003171. 1878
2. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, Nisalak A, Yoon IK, 1879 Fernandez S. Evaluation of a Dengue NS1 antigen detection assay sensitivity and specificity for the diagnosis 1880 of acute Dengue virus infection. PLoSNegl Trop Dis. 2014 Oct 2;8(10):e3193. doi: 1881 10.1371/journal.pntd.0003193. 1882
3. Yow KS, Aik J, Tan EY, Ng LC, Lai YL. Rapid diagnostic tests for the detection of recent Dengue infections: 1883 An evaluation of six kits on clinical specimens. PLoS One. 2021 Apr 1;16(4):e0249602. doi: 1884 10.1371/journal.pone.0249602. 1885

stic tests for the detection of recent Dengue infections: 1883 An evaluation of six kits on clinical specimens. PLoS One. 2021 Apr 1;16(4):e0249602. doi: 1884 10.1371/journal.pone.0249602. 1885

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 83 | Page

4. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – Diagnostic 1886 Assessment TGS-3. 2017. Available at: https://iris.who.int/bitstream/handle/10665/258985/WHO-EMP- 1887 RHT-PQT-TGS3-2017.03-eng.pdf;sequence=1
   1888
5. WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. (Diagnostics 1889 evaluation series, 3). ISBN 978 92 4 159775 3.
   1890

1891 VII. 1892

1893

1894

1895

1896

1897

1898

1899

1900

1901

1902

1903

1904

1905

1906

1907

1908

1909

1910

1911

1912

1913

1914

1915

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 84 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE NS1 and IgM COMBO RDT 1916 KIT 1917

1918 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No.

REPORT FOR DENGUE NS1 and IgM COMBO RDT 1916 KIT 1917

1918 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: strong, moderate, weak)
Negative samples (provide details: clinical/spiked, including cross reactivity panel)

1919 Results: 1920

Positive Negative Total Name of Dengue NS1 and IgM combo RDT kit Positive

Negative

Total

1921

Estimate (%) 95% CI Combined Sensitivity

Combined Specificity

1922 ● Details of cross reactivity with other flavivirus NS1 antigens:
1923 ● Conclusions: 1924

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 85 | Page

o Concordance for NS1, Concordance for IgM 1925 o Sensitivity, specificity 1926 o 1927 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 1928 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 1929 Disclaimers 1930 1931 1932

1933 Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by …………… (Supplied by ……….) 1934 1935 1936 Signature of Director/ Director-In-charge …………………… Seal ……………………………… 1937 ****End of the Report 1938

1939

1940

1941

1942

1943

1944

1945

1946

1947

1948

1949

1950

1951

1952

1953

1954

1955

1956

End of the Report************************* 1938

1939

1940

1941

1942

1943

1944

1945

1946

1947

1948

1949

1950

1951

1952

1953

1954

1955

1956

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 86 | Page

Field evaluation protocol for Dengue NS1 and IgM combo RDT kits 1957 I. Background:
1958 1959 1960 1961 1962 II. Purpose: 1963 To evaluate the performance characteristics of Dengue NS1/IgM RDT combo kits in the diagnosis 1964 of Dengue infection in individuals with unknown disease status. 1965 III. Requirements:
1966 1967 1968 1969 1970 1971 4. Laboratory supplies
1972

1973 IV. Ethical approval:
1974 The study will be initiated after approval from the institutional human ethics committee.
1975 V. Procedure: 1976

1. Study design/type: Cross-sectional study 1977 1978 1979 1980 1981 1982 1983 1984 1985 ⮚ Handling of Dengue NS1 RDT/IgM RDT IVD kits received for performance evaluation 1986 1987 1988 1989

dy design/type: Cross-sectional study 1977 1978 1979 1980 1981 1982 1983 1984 1985 ⮚ Handling of Dengue NS1 RDT/IgM RDT IVD kits received for performance evaluation 1986 1987 1988 1989

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 87 | Page

3. Sample size for performance evaluation: 1990 Sample sizes of positive and negative samples of Dengue against different values of 1991 sensitivity and specificity are provided in Tables 1 and 2. Sample sizes have been calculated 1992 assuming 95% level of significance, an absolute precision of 5%, and invalid test rate ≤5%. 1993 It is further assumed that 30% of the individuals attending the health care facilities for acute 1994 febrile illness and suspected for Dengue will be positive for Dengue. Appropriate sample 1995 size has to be chosen from the tables according to the values of sensitivity and specificity 1996 being claimed by the manufacturer. If a claimed sensitivity/specificity is not present in the 1997 table, the manufacturer needs to consider the sample size associated with the largest 1998 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as 1999 per the next smaller value of the sensitivity/ specificity available in the table). For example, 2000 if a manufacturer claims a sensitivity of 93%, they are required to use a sample size 2001 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 2002 usage of the sample size outlined for 85% specificity.

93%, they are required to use a sample size 2001 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 2002 usage of the sample size outlined for 85% specificity.
2003 Sample size has to be calculated based on both the sensitivity and the specificity. The final 2004 sample size will be the maximum of the two. For example, at 95% sensitivity and 95% 2005 specificity, the sample size required will be 260 (maximum of 260 and 110). It is desirable 2006 to cover at least one Dengue season so that adequate samples are available for evaluation. 2007

2008 Table 1. Sample sizes for different values of sensitivity claimed by the manufacturer. 2009 Sensitivity Calculated sample size No. of individuals* off] 99%# 53 60 95% 255 260 90% 484 490 85% 686 690 80% 861 870

• Individuals attending the health care facilities for

2010 #Higher sample size should be used even for assays claiming 99% sensitivity. 2011

2012 Table 2. Sample sizes for different values of specificity claimed by the manufacturer. 2013 Specificity Calculated sample size No. of individuals* off] 99%# 23 30 95% 109 110 90% 207 210 85% 294 300

zes for different values of specificity claimed by the manufacturer. 2013 Specificity Calculated sample size No. of individuals* off] 99%# 23 30 95% 109 110 90% 207 210 85% 294 300

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 88 | Page

80% 369 370

• Individuals attending the health care facilities for

2014 #Higher sample size should be used even for assays claiming 99% specificity. 2015 Recruitment of cases shall be halted once desired number of positive and negative samples are 2016 reached. 2017

2018 4. Inclusion criteria: 2019 Patient with Dengue like illness (A patient with acute febrile illness of 1-14 days with two or more 2020 manifestations: Head ache, retro-orbital pain, myalgia, arthralgia, rash, hemorrhagic 2021 manifestations etc. The 1-14 days disease duration shall cover viraemic as well as convalescent 2022 phase of Dengue infection, so that both Dengue NS1 and IgM positive cases are enrolled.) 2023 5. Exclusion criteria: 2024 Individuals with already known positive history for other pathogens 2025 6. Reference assay:
2026 Anti-DENV IgM detection ELISA US-FDA approved kit 2027 AND/OR 2028 DENV NS1 ELISA US-FDA approved kit 2029 Serotype status to be assessed using a combination of CDC and/or NIV real-time PCR serotyping 2030 protocols.
2031 7. Study implementation: 2032 The individuals with Dengue like illness will be recruited into the study and five ml of whole blood 2033 will be collected in vacutainer tubes and the serum will be separated by centrifugation and used 2034 for the study.

like illness will be recruited into the study and five ml of whole blood 2033 will be collected in vacutainer tubes and the serum will be separated by centrifugation and used 2034 for the study.
2035 It needs to be ensured that the samples are tested by reference tests and index test simultaneously. 2036 8. Positive samples: 2037 Samples which are positive for IgM or NS1 or both by the reference assays will be considered as 2038 true positive samples.
2039 9. Negative samples:
2040 Samples which are negative by the reference assay will be considered as negative. 2041 A. Cross reactivity (other flavivirus infections): 2042

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 89 | Page

A.1 NS1: 2043 Clinical samples or commercially available NS1 antigens from other flaviviruses will be used 2044 to test cross reactivity of the NS1 component of index test. 2045 i. Japanese Encephalitis PCR/antigen positive: 5 samples* 2046 ii. West Nile Virus PCR/antigen: 5 samples* 2047 iii. Zika Virus PCR/antigen: 5 samples* 2048 *In the absence of natural samples, spiked samples may be used, as per details provided in the note below. 2049 Note:
2050 Recombinant NS1 antigen of cross reactive flaviviruses (Zika, West Nile and Japanese Encephalitis viruses) expressed 2051 in mammalian cells can be obtained commercially and reconstituted in serum samples (100 ng -1 µg/ml) and diluted 2052 in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1).

ls can be obtained commercially and reconstituted in serum samples (100 ng -1 µg/ml) and diluted 2052 in the ratio of 1:2 and used accordingly (at least five dilutions for each virus specific NS1). 2053 Before used for evaluation, NS1 reconstituted in serum samples needs to be tested by the reference assay and dilution 2054 which are positive only should be used for evaluation. 2055 The serum samples used for reconstitution should be negative for Dengue NS1, RNA and IgM antibody. 2056 A.2 IgM: 2057 Clinical samples positive for IgM for other flaviviruses will be used to test cross reactivity of 2058 the IgM component of index test. 2059 i. Japanese Encephalitis IgM positive: 5 samples 2060 ii. West Nile Virus IgM positive: 5 samples 2061 iii. Zika Virus IgM positive: 5 samples 2062 Note: Depending on the availability of IgM positive samples for cross reactive flaviviruses, the requirement of samples 2063 for each virus may be increased or decreased accordingly to reach the total number of samples. If IgM positive samples 2064 for cross reactive flaviviruses are not available, commercially available IgM sera panel for different viruses can be 2065 procured and used to test cross reactivity. 2066 10. Statistical analysis:
2067 Concordance will be calculated separately for Dengue NS1 and IgM. Combined sensitivity and 2068 specificity will also be calculated. 2069 Interim analysis of data shall be conducted on completing evaluation of 25%, 50% and 75% of 2070 samples.

ue NS1 and IgM. Combined sensitivity and 2068 specificity will also be calculated. 2069 Interim analysis of data shall be conducted on completing evaluation of 25%, 50% and 75% of 2070 samples. If, at any point, the performance of the assay is found to be not satisfactory, the assay 2071 shall not be evaluated further. Evaluation fee shall be charged accordingly. 2072

2073 11. Test reproducibility 2074 2075 2076 as follows:
2077 2078 2079

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 90 | Page

2080 2081 samples).
2082 2083 2084

2085 2086

2087 B. Sample size for reader-to-reader reproducibility 2088 For reader-to-reader reproducibility, sample size should be 25 (15 positive samples comprising 10 2089 low positive AND 5 moderate/high positive samples with adequate representation of NS1 and 2090 IgM, and 10 negative samples). 2091

2092 Two operators will be reading the test results independently as per manufacturer’s instruction. 2093 Agreement should be 100% between the operators. 2094 C. Interpretation of results
2095 Since the kits have been provided in a combo format, the sensitivity and specificity has to be 2096 calculated based on the combined results of the NS1 and IgM. If the sample is positive for any one 2097 or both analytes (NS1 or IgM or both), then the sample is considered positive. Refer the table 2098 below: 2099

e combined results of the NS1 and IgM. If the sample is positive for any one 2097 or both analytes (NS1 or IgM or both), then the sample is considered positive. Refer the table 2098 below: 2099

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 91 | Page

NS1 Reference test result IgM reference test result Final Reference test result NS1 Index test result IgM Index test result Final index test result Interpretation + + Positive +

Positive True Positive + + Positive

Positive True Positive + + Positive

Negative False Negative + + Positive + + Positive True Positive +

Positive +

Positive True Positive +

Positive

Positive True Positive +

Positive

Negative False Negative

Positive +

Positive True Positive

Positive

Positive True Positive

Positive

Negative False Negative

Negative

Positive False Positive

Negative +

Positive False Positive

2100 12. Acceptance criteria: 2101 A minimum concordance of 80% for NS1 and 80% for IgM should be achieved with the reference 2102 assay, and an overall combined sensitivity* and specificity$ of ≥90% each.

cceptance criteria: 2101 A minimum concordance of 80% for NS1 and 80% for IgM should be achieved with the reference 2102 assay, and an overall combined sensitivity* and specificity$ of ≥90% each. 2103 Cross reactivity with other flavivirus antigens: Nil 2104 2105

• Samples which are positive for NS1 or IgM or both by the kit under evaluation (index test) irrespective 2106 of the reference assay results will be considered as positive and used for sensitivity calculation 2107 $ Samples which are negative for both NS1 and IgM by kit under evaluation only will be considered as 2108 negative and used for specificity calculation 2109 13. Publication Rights: 2110 2111

2112 2113 2114 2115 2116

2117

2118 VI. References: 2119

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian 2120 E, Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, 2121

19

1. Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA, Vazquez S, Cartozian 2120 E, Pelegrino JL, Artsob H, Guzman MG, Olliaro P, Zwang J, Guillerm M, Kliks S, Halstead S, 2121

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 92 | Page

Peeling RW, Margolis HS. Evaluation of commercially available diagnostic tests for the detection 2122 of Dengue virus NS1 antigen and anti-Dengue virus IgM antibody. PLoSNegl Trop Dis. 2014 Oct 2123 16;8(10):e3171. doi: 10.1371/journal.pntd.0003171. 2124 2. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, 2125 Nisalak A, Yoon IK, Fernandez S. Evaluation of a Dengue NS1 antigen detection assay 2126 sensitivity and specificity for the diagnosis of acute Dengue virus infection. PLoSNegl 2127 Trop Dis. 2014 Oct 2;8(10):e3193. doi: 10.1371/journal.pntd.0003193. 2128 3. Ganeshkumar P, Murhekar MV, Poornima V, Saravanakumar V, Sukumaran K, 2129 Anandaselvasankar A, John D, Mehendale SM. Dengue infection in India: A systematic 2130 review and meta-analysis. PLoSNegl Trop Dis. 2018 Jul 16;12(7):e0006618. doi: 2131 10.1371/journal.pntd.0006618. 2132 4. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 2133 Diagnostic Assessment TGS-3. 2017. Available at: 2134 2135 eng.pdf;sequence=1
2136 5. WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. 2137 (Diagnostics evaluation series, 3). ISBN 978 92 4 159775 3.
2138

2139

2140 VII.

uence=1
2136 5. WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. 2137 (Diagnostics evaluation series, 3). ISBN 978 92 4 159775 3.
2138

2139

2140 VII. 2141

2142

2143

2144

2145

2146

2147

2148

2149

2150

2151

2152

2153

2154

2155

2156

2157

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 93 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE NS1 and IgM COMBO RDT 2158 KIT 2159 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples: Not applicable, may categorize cases as per duration of illness Negative samples (may categorize as per duration of illness, must include cross reactivity panel) Results 2160

Positive Negative Total Name of NS1 and IgM combo RDT kit Positive

Negative

Total

2161

Estimate (%) 95% CI Sensitivity

Specificity

2162 ● Details of cross reactivity with other flavivirus NS1 antigens:
2163 ● Conclusions: 2164 o Sensitivity, specificity 2165

Estimate (%) 95% CI Sensitivity

Specificity

2162 ● Details of cross reactivity with other flavivirus NS1 antigens:
2163 ● Conclusions: 2164 o Sensitivity, specificity 2165

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 94 | Page

o 2166 (Sensitivity and specificity have been assessed in using kits provided by the manufacturer from the batch mentioned above using 2167 ….. sample in ……. (field/controlled lab). Results should not be extrapolated to other sample types.) 2168 Disclaimers 2169 2170 2171 Note: This report is exclusively for …………………..NS1 and IgM combo Kit (Lot No……) manufactured by 2172 …………… (supplied by ……….) 2173 2174 2175 Signature of Director/ Director-In charge …………………… Seal ……………………………… 2176 ****End of the Report 2177

2178

2179

2180

2181

2182

2183

2184

2185

2186

2187

2188

2189

2190

2191

2192

2193

2194

2195

2196

f the Report**************************** 2177

2178

2179

2180

2181

2182

2183

2184

2185

2186

2187

2188

2189

2190

2191

2192

2193

2194

2195

2196

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 95 | Page

Performance evaluation protocol for Dengue real-time PCR kit 2197 I. Background:
2198 2199 2200 2201 2202 This recommendation focuses on the laboratory performance evaluation of Dengue virus 2203 molecular diagnostic test. All clinical samples tested in the study should be evaluated in 2204 accordance with the candidate test’s instructions for use.
2205

2206 II. Purpose: 2207 To evaluate the performance characteristics of Dengue real-time PCR kits in the diagnosis of 2208 Dengue infection. 2209 III. Requirements:
2210 2211 2212 2213 2214 2215 2216 2217 IV. Ethical approvals:
2218 2219 2220 2221 V. Procedure: 2222

1. Study design/type: Diagnostic accuracy study using archived/ leftover/ spiked clinical 2223 samples. 2224 2225 2226 A. Accreditation form NABL for at least one of the Quality management system (NABL 2227 2228 2229

ostic accuracy study using archived/ leftover/ spiked clinical 2223 samples. 2224 2225 2226 A. Accreditation form NABL for at least one of the Quality management system (NABL 2227 2228 2229

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 96 | Page

B. Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 2230 2231 2232 ⮚ Handling of Dengue RT-PCR kits received for performance evaluation 2233 2234 2235 2236 3. Preparation of Dengue RNA evaluation panel 2237 Well characterised Dengue serum/plasma panel positive for RNA by RT-PCR is a critical 2238 requirement for performance evaluation of IVD kits utilizing genome detection. Hence statistically 2239 significant number of sera/plasma samples should be available from Dengue PCR confirmed cases.
2240 4. RNA extraction 2241 RNA extraction shall be performed using standard techniques. If the manufacturer of the index test 2242 recommends a specific RNA extraction kit, the same needs to be provided by the manufacturer.
2243 5. Real-Time PCR System 2244 PCR shall be performed using IVD-approved machines. If any equipment(s) is specified in the 2245 IFU of the index test, it shall be used for the evaluation, and it shall be provided by the 2246 manufacturer if not available within the lab’s IVD evaluation scope.
2247 6. Internal control/Extraction control 2248 The test under evaluation should have an internal control or extraction control (RNA added before 2249 extraction to a sample). 2250 7.

cope.
2247 6. Internal control/Extraction control 2248 The test under evaluation should have an internal control or extraction control (RNA added before 2249 extraction to a sample). 2250 7. Reference assay:
2251 Any FDA approved Dengue PCR assay or CDC/NIV protocol for detection of Dengue virus RNA 2252 should be used as the reference assay. 2253 All positive samples should be confirmed positive for at least one serotype by real-time PCR assay 2254 using CDC/NIV protocol.
2255 All negative samples should be negative for all the markers of Dengue infection (NS1, IgM, and 2256 RNA).
2257

2258 8. Sample size and sample panel composition: Sample sizes of positive and negative 2259 samples and sample panel composition against different values of sensitivity and specificity are 2260 provided in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of significance, 2261

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 97 | Page

an absolute precision of 5%, and invalid test rate ≤5%. Appropriate sample size has to be chosen 2262 from the tables according to the values of sensitivity and specificity being claimed by the 2263 manufacturer. If a claimed sensitivity/specificity is not present in the table, the manufacturer needs 2264 to consider the sample size associated with the largest sensitivity/specificity provided in the table 2265 that is smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ specificity 2266 available in the table).

rgest sensitivity/specificity provided in the table 2265 that is smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ specificity 2266 available in the table). For example, if a manufacturer claims a sensitivity of 93%, they are required 2267 to use a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would 2268 require usage of the sample size outlined for 85% specificity.
2269 Positive samples: These include samples positive by the reference real-time PCR assay (True 2270 positives) and representative of all four serotypes.
2271

2272 Negative samples: All negative samples should be negative by reference real-time PCR assay, US- 2273 FDA approved NS1 antigen ELISA kit-and US FDA approved IgM Capture ELISA.
2274

2275

2276 Table 1. Sample sizes and panel composition of positive Dengue samples for different values of 2277 sensitivity claimed by the manufacturer. 2278 Sensitivity Calculated sample size No. of Positive Samples required off] 99%# 16 20 Strong positive (Ct value <25): 5 Moderate positive (Ct value between 25-30): 10
Weak positive (Ct value >30 to 34): 5
95% 77 80 20
between 25-30): 40
34): 20 90% 145 150 38
between 25-30): 74
34): 38 85% 206 210 53

positive (Ct value between 25-30): 10
Weak positive (Ct value >30 to 34): 5
95% 77 80 20
between 25-30): 40
34): 20 90% 145 150 38
between 25-30): 74
34): 38 85% 206 210 53

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 98 | Page

between 25-30): 104
34): 53 80% 258 260 65
between 25-30): 130
34): 65 Note: If clinical samples positive for a particular serotype is not available, tissue culture fluid (5-10 different isolates with a plaque forming unit of 105-6/ml) (Heat-inactivated) from reference laboratories can be obtained, spiked in serum samples (15 µl isolate + 150 µl) and can be further diluted in the ratio of 1:10, frozen at -80°C, and tested by the reference assay when needed and the positive samples can be used for evaluation.
The serum used for spiking isolate should be negative for Dengue virus RNA, and NS1.

2279 #Higher sample size should be used even for assays claiming 99% sensitivity. 2280

2281 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 2282 specificity claimed by the manufacturer. 2283 Specificity Calculated sample size No.

2281 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 2282 specificity claimed by the manufacturer. 2283 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off] 99%# 16 20 Chikungunya positive: 4
aAcute febrile cases: 8
*Japanese Encephalitis positive: 1
*West Nile Virus positive: 1 *Zika Virus positive: 1 bHealthy subjects from endemic regions: 5
95% 77 80 Chikungunya positive: 15
aAcute febrile cases: 30
*Japanese Encephalitis positive: 5
*West Nile Virus positive: 5 *Zika Virus positive: 5 bHealthy subjects from endemic regions: 20
90% 145 150 Chikungunya positive: 28
aAcute febrile cases: 57
*Japanese Encephalitis positive: 9
*West Nile Virus positive: 9 *Zika Virus positive: 9

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 99 | Page

bHealthy subjects from endemic regions: 38
85% 206 210 Chikungunya positive: 39
aAcute febrile cases: 79
*Japanese Encephalitis positive: 13
*West Nile Virus positive: 13 *Zika Virus positive: 13 bHealthy subjects from endemic regions: 53
80% 258 260 Chikungunya positive: 49
aAcute febrile cases: 98
*Japanese Encephalitis positive: 16
*West Nile Virus positive: 16 *Zika Virus positive: 16 bHealthy subjects from endemic regions: 65
a Acute febrile cases negative for all markers of Dengue (NS1 & IgM & IgG & RNA) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid)

• Note:

rile cases negative for all markers of Dengue (NS1 & IgM & IgG & RNA) b Samples from healthy subjects from endemic regions negative for all Dengue markers (NS1, IgM, IgG, nucleic acid)

• Note:
  If PCR positive samples for cross reactive flaviviruses not available, commercially available RNA panels should be used to test cross reactivity.

2284 #Higher sample size should be used even for assays claiming 99% specificity. 2285 9. Evaluation method: 2286 The index test and the reference tests should be run simultaneously on the sample panel to 2287 avoid false negative results by index test due to free thawing of samples or deterioration of 2288 sample quality on long term storage. Both the index and reference tests should be run on 2289 the sample plate for each of the panel samples. 2290 10. Test reproducibility 2291 2292 2293 as follows:
2294 2295 2296 2297 2298 samples).
2299 2300 2301

2302 2303

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 100 | Page

2304

2305

2306 11. Acceptance Criteria 2307 Expected sensitivity: ≥95% 2308 Expected specificity: ≥98% 2309 Cross reactivity with other flavivirus: Nil 2310 2311

2312 13. Publication Rights: 2313 2314

2315 2316 2317 2318 2319

2320 VI. References: 2321

ted specificity: ≥98% 2309 Cross reactivity with other flavivirus: Nil 2310 2311

2312 13. Publication Rights: 2313 2314

2315 2316 2317 2318 2319

2320 VI. References: 2321

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 101 | Page

1. Santiago, G.A., Vázquez, J., Courtney, S. et al. Performance of the Trioplex real-time RT-PCR assay 2322 for detection of Zika, Dengue, and Chikungunya viruses. Nat Commun 9, 1391 (2018). 2323 https://doi.org/10.1038/s41467-018-03772-1 2324
2. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 2325 Diagnostic Assessment TGS-3.

Available at: 2326 2327 eng.pdf;sequence=1
2328

2329 VII. 2330

2331

2332

2333

2334

2335

2336

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2338

2339

2340

2341

2342

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ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 102 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE REAL-TIME PCR KITS 2353 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No.

PERFORMANCE EVALUATION REPORT FOR DENGUE REAL-TIME PCR KITS 2353 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: clinical/ spiked, strong, moderate, weak)
Negative samples (provide details clinical/ spiked, including cross reactivity panel)

2354 Results 2355

Positive Negative Total Name of Dengue real- time PCR Positive

Negative

Total

2356

Estimate (%) 95% CI Sensitivity

Specificity

2357 ● Details of cross reactivity with other flaviviruses: 2358 ● Conclusions: 2359 o Sensitivity, specificity 2360

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 103 | Page

o Performance: Satisfactory / Not satisfactory 2361 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 2362 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 2363 Disclaimers 2364 2365 2366 Note: This report is exclusively for Dengue………….. Kit (Lot No……) manufactured by …………… 2367 (supplied by ……….) 2368 2369 2370 Signature of Director/ Director-In-charge …………………… Seal …………………………. 2371 ****End of the Report 2372

2373

2374

2375

2376

2377

2378

2379

2380

2381

2382

2383

2384

2385

2386

2387

2388

2389

2390

End of the Report************************* 2372

2373

2374

2375

2376

2377

2378

2379

2380

2381

2382

2383

2384

2385

2386

2387

2388

2389

2390

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 104 | Page

Field evaluation protocol for Dengue real-time PCR kits 2391 I. Background:
2392 2393 2394 2395 2396 II. Purpose: 2397 To evaluate the performance characteristics of Dengue real-time PCR kits in the diagnosis of 2398 Dengue infection in individuals with unknown disease status. 2399 III. Requirements:
2400 2401 2402 2403 2404 2405 4. Laboratory supplies
2406

2407 IV. Ethical approvals:
2408 The study will be initiated after approval from the institutional human ethics committee.
2409 V. Procedure: 2410

1. Study design/type: Cross-sectional study 2411 2412 2413 A. Accreditation form NABL for at least one of the Quality management system (NABL 2414 2415 2416 B. Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 2417 2418 2419 ⮚ Handling of Dengue RT-PCR kits received for performance evaluation 2420 2421 2422 2423

All the staff involved in IVD kit evaluation should undergo hands on training 2417 2418 2419 ⮚ Handling of Dengue RT-PCR kits received for performance evaluation 2420 2421 2422 2423

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 105 | Page

3. Sample size for performance evaluation: 2424 Sample sizes of positive and negative samples of Dengue against different values of 2425 sensitivity and specificity are provided in Tables 1 and 2. Sample sizes have been calculated 2426 assuming 95% level of significance, an absolute precision of 5%, and invalid test rate ≤5%. 2427 It is further assumed that 30% of the individuals attending the health care facilities for acute 2428 febrile illness and suspected for Dengue will be positive for Dengue. Appropriate sample 2429 size has to be chosen from the tables according to the values of sensitivity and specificity 2430 being claimed by the manufacturer. If a claimed sensitivity/specificity is not present in the 2431 table, the manufacturer needs to consider the sample size associated with the largest 2432 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as 2433 per the next smaller value of the sensitivity/ specificity available in the table). For example, 2434 if a manufacturer claims a sensitivity of 93%, they are required to use a sample size 2435 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 2436 usage of the sample size outlined for 85% specificity.

93%, they are required to use a sample size 2435 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 2436 usage of the sample size outlined for 85% specificity.
2437 Sample size has to be determined based on both the sensitivity and the specificity. The 2438 required sample size will be the maximum of the two. For example, at 95% sensitivity 2439 and 95% specificity, the sample size required will be 260 (maximum of 260 and 110). 2440

2441 Table 1. Sample sizes for different values of sensitivity claimed by the manufacturer. 2442 Sensitivity Calculated sample size No. of individuals* off] 99%# 53 60 95% 255 260 90% 484 490 85% 686 690 80% 861 870

• Individuals attending the health care facilities for

2443 #Higher sample size should be used even for assays claiming 99% sensitivity. 2444

2445 Table 2. Sample sizes for different values of specificity claimed by the manufacturer. 2446 Specificity Calculated sample size No. of individuals* off] 99%# 23 30 95% 109 110 90% 207 210 85% 294 300 80% 369 370

nt values of specificity claimed by the manufacturer. 2446 Specificity Calculated sample size No. of individuals* off] 99%# 23 30 95% 109 110 90% 207 210 85% 294 300 80% 369 370

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 106 | Page

• Individuals attending the health care facilities for

2447 #Higher sample size should be used even for assays claiming 99% specificity. 2448 Recruitment of cases shall be halted once desired number of positive and negative samples are 2449 reached. 2450 4. Inclusion criteria: 2451 Individuals with Dengue like illness (A patient with acute febrile illness of 2-7 days with two or 2452 more manifestations: Head ache, retro-orbital pain, myalgia, arthralgia, rash, hemorrhagic 2453 manifestations) 2454 5. Exclusion criteria: 2455 Individuals with already known positive history for other pathogens 2456 6. RNA extraction 2457 RNA extraction shall be performed using standard techniques. If any extraction system is specified
2458 in the IFU, that shall be used for the test and shall be provided by the manufacturer.
2459 7. Real-Time PCR System 2460 PCR shall be performed using IVD-approved machines. If any equipment(s) is specified in the 2461 IFU, that shall be used for the test and shall be provided by the manufacturer.
2462 8. Internal control/Extraction control 2463 The test under evaluation should have an internal control or extraction control (RNA added before 2464 extraction to a sample). 2465 9.

urer.
2462 8. Internal control/Extraction control 2463 The test under evaluation should have an internal control or extraction control (RNA added before 2464 extraction to a sample). 2465 9. Reference assay:
2466 Any FDA approved Dengue PCR assay or CDC/NIV protocol for detection of Dengue RNA 2467 should be used as the reference assay. 2468 All positive samples should be confirmed positive for at least one serotype by real-time PCR assay 2469 using CDC/NIV protocol.
2470 All negative samples should be negative for all the markers of Dengue infection (NS1 & IgM & 2471 IgG and RNA).
2472 10. Study implementation: 2473 The individuals with Dengue like illness will be recruited into the study and five ml of whole blood 2474 will be collected in vacutainer tubes and the serum will be separated by centrifugation and used 2475 for the study.
2476

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 107 | Page

It needs to be ensured that the samples are tested by reference tests and index test simultaneously. 2477 11. Positive samples: 2478 Samples which are positive by reference real-time PCR assay will be considered as true positive 2479 sample.
2480 12. Negative samples:
2481 Samples which are negative by the reference assay will be considered as negative. 2482 A. Cross reactivity: 2483 Clinical samples or commercially available Viral RNA genome of other flaviviruses/RNA from 2484 sequence confirmed virus isolates will be used to test cross reactivity of the index test. 2485 a. Japanese Encephalitis PCR positive: 5 samples 2486 b.

nome of other flaviviruses/RNA from 2484 sequence confirmed virus isolates will be used to test cross reactivity of the index test. 2485 a. Japanese Encephalitis PCR positive: 5 samples 2486 b. West Nile Virus PCR positive: 5 samples 2487 c. Zika Virus PCR positive: 5 samples 2488 Alternatively, tissue culture fluid of cross reactive flaviviruses (with a plaque forming unit of 105-6/ml)(Heat 2489 inactivated) from reference laboratories can be obtained, spiked in serum samples (15 µl isolate + 150 µl) and can be 2490 further diluted in the ratio of 1:10, tested by the reference assay and the negative samples can be used for evaluation. 2491 The serum used for spiking isolate should be negative for Dengue virus RNA, and NS1. 2492 13. Statistical analysis:
2493 Sensitivity and specificity will be calculated. 2494 Interim analysis of data shall be conducted on completing evaluation of 25%, 50% and 75% of 2495 samples. If, at any point, the performance of the assay is found to be not satisfactory, the assay 2496 shall not be evaluated further. Evaluation fee shall be charged accordingly. 2497 14. Test reproducibility 2498 2499 2500 as follows:
2501 2502 2503 2504 2505 samples).
2506 2507 2508

2509 2510

luated further. Evaluation fee shall be charged accordingly. 2497 14. Test reproducibility 2498 2499 2500 as follows:
2501 2502 2503 2504 2505 samples).
2506 2507 2508

2509 2510

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 108 | Page

2511 15. Acceptance Criteria 2512 Sensitivity: ≥95% 2513 Specificity: ≥98% 2514 Cross reactivity with other flavivirus: Nil 2515 2516 16. Publication Rights: 2517 2518

2519 2520 2521 2522 2523

2524 VI. References: 2525

1. Santiago, G.A., Vázquez, J., Courtney, S. et al. Performance of the Trioplex real-time RT-PCR 2526 assay for detection of Zika, Dengue, and Chikungunya viruses. Nat Commun 9, 1391(2018). 2527 https://doi.org/10.1038/s41467-018-03772-1 2528
2. Ganeshkumar P, Murhekar MV, Poornima V, Saravanakumar V, Sukumaran K, 2529 Anandaselvasankar A, John D, Mehendale SM. Dengue infection in India: A systematic 2530

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 109 | Page

review and meta-analysis. PLoSNegl Trop Dis. 2018 Jul 16;12(7):e0006618. doi: 2531 10.1371/journal.pntd.0006618. 2532 3. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 2533 Diagnostic Assessment TGS-3. 2017. Available at: 2534 2535 eng.pdf;sequence=1
2536

2537 VII. 2538

2539

2540

2541

2542

2543

2544

2545

2546

2547

2548

2549

2550

2551

2552

2553

2554

2555

2556

2557

2558

2559

2560

2561

37 VII. 2538

2539

2540

2541

2542

2543

2544

2545

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2558

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2560

2561

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 110 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE REAL-TIME PCR KITS 2562 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples: Not applicable, may categorize cases as per duration of illness Negative samples (may categorize as per duration of illness, must include cross reactivity panel)

2563 Results 2564

Positive Negative Total Name of Dengue real-time PCR kit Positive

Negative

Total

2565

Estimate (%) 95% CI Sensitivity

Specificity

2566 ● Details of cross reactivity with other flaviviruses: 2567 ● Conclusions: 2568 o Sensitivity, specificity 2569

2565

Estimate (%) 95% CI Sensitivity

Specificity

2566 ● Details of cross reactivity with other flaviviruses: 2567 ● Conclusions: 2568 o Sensitivity, specificity 2569

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 111 | Page

o Performance: Satisfactory / Not satisfactory 2570 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 2571 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 2572 Disclaimers 2573 2574 2575

2576 Note: This report is exclusively for Dengue………….. Kit (Lot No……) manufactured by …………… (supplied by 2577 ……….) 2578 2579 2580 Signature of Director/ Director-In-charge …………………… Seal ……………………………….. 2581 ****End of the Report 2582

2583

2584

2585

2586

2587

2588

2589

2590

2591

2592

2593

2594

2595

2596

2597

End of the Report* 2582

2583

2584

2585

2586

2587

2588

2589

2590

2591

2592

2593

2594

2595

2596

2597

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 112 | Page

Performance evaluation protocol for Real-time PCR tests for Zika virus 2598 I. Background:
2599 CDSCO and ICMR, New Delhi, aimed at facilitating the evaluation and deployment of Quality- 2600 Assured Diagnostics kits appropriate for use in India. Hence the following guidelines shall 2601 establish the uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The 2602 performance evaluation is to independently verify the manufacturer’s claim regarding in-vitro 2603 diagnostic kit (IVD) performance. 2604 This recommendation focuses on the laboratory performance evaluation of Zika virus molecular 2605 diagnostic test. All clinical samples tested in the study should be evaluated in accordance with the 2606 candidate test’s proposed diagnostic algorithm (i.e., tested using the procedure in the instructions 2607 for use), including retesting when appropriate.
2608 II. Purpose: To evaluate the performance characteristics of Zika virus RT-PCR test for diagnosis 2609 of Zika infection.
2610 III. Requirements:
2611

1. Supply of kits under evaluation (along with batch/lot No. Expiry & required details) 2612
2. Evaluation site/laboratory should be equipped with necessary equipment and supplies for 2613 molecular testing.

kits under evaluation (along with batch/lot No. Expiry & required details) 2612 2. Evaluation site/laboratory should be equipped with necessary equipment and supplies for 2613 molecular testing. Any essential equipment and consumables for closed system to be 2614 supplied and maintained from the manufacturer, during the period of evaluation. 2615 2616 4. Characterized evaluation panel 2617 2618 IV. Ethics approval: Exempted from Ethics approval as per ICMR’s Guidance on Ethical 2619 Requirements for Laboratory Validation Testing, 2024. A self-declaration form as provided in 2620 ICMR guidelines to be submitted by the investigators to the institutional authorities and ethics 2621 committee for information. 2622 V. Procedure:
2623

1. Study design: Diagnostic accuracy study using archived/leftover/spiked clinical samples. 2624
2. Preparation of Evaluation site/laboratory: Performance evaluation performance and report 2625 to be issued only from designated reference testing laboratory/ NABL accredited laboratory, as 2626 specified by state or central licensing authority. 2627
3. Identified IVD kit evaluation laboratories should establish their proficiency through
   2628

atory/ NABL accredited laboratory, as 2626 specified by state or central licensing authority. 2627 3. Identified IVD kit evaluation laboratories should establish their proficiency through
2628

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 113 | Page

A.NABL accreditation for at least one of the Quality management system (NABL accreditation 2629 for testing laboratory/ calibration laboratory (ISO/IES 17025), Medical Laboratory (ISO 2630 15189), PT provider ISO/IEC 17043 or CDSCO approved Reference laboratory. 2631 B.Staff training: All the staff involved in the IVD kit evaluation should undergo hands on 2632 training and competency testing on following
2633 ⮚ Preparation & characterization of evaluation panel
2634 ⮚ Handling of Zika molecular diagnostic kits received for performance evaluation 2635 (Verification/Storage/Unpacking etc.) 2636 ⮚ Testing, interpretation, recording of results & reporting 2637 2638

2639 4. Preparation of Zika reference evaluation panel 2640 Well characterized Zika molecular evaluation panel is a critical requirement for performance 2641 evaluation of IVD kits. Hence, statistically significant number of clinical samples should be used 2642 for evaluation. 2643 ● Frozen samples (≤-70°C) may be used, if stored appropriately and analytical data 2644 demonstrate that accuracy of test results is not affected.
2645 ● Samples that previously tested positive by FDA approved PCR and/or CDC/NIV 2646 approved protocols may be used. 2647 ● In the absence of natural samples, spiked clinical samples may be used.

5 ● Samples that previously tested positive by FDA approved PCR and/or CDC/NIV 2646 approved protocols may be used. 2647 ● In the absence of natural samples, spiked clinical samples may be used. 2648 5. RNA extraction 2649 RNA extraction shall be performed using standard techniques. If the manufacturer of the index test 2650 recommends a specific RNA extraction kit, the same needs to be provided by the manufacturer.
2651 6. Real-Time PCR System 2652 PCR shall be performed using IVD-approved machines. If any equipment(s) is specified in the 2653 IFU of the index test, it shall be used for the evaluation, and it shall be provided by the 2654 manufacturer if not available within the lab’s IVD evaluation scope.
2655 7. Internal control/Extraction control 2656 The test under evaluation should have an internal control or extraction control (RNA added before 2657 extraction to a sample). 2658 8. Reference assay:
2659 Any FDA approved Zika PCR assay or CDC/NIV protocol for detection of Zika RNA should be 2660 used as the reference assay. 2661

e 2657 extraction to a sample). 2658 8. Reference assay:
2659 Any FDA approved Zika PCR assay or CDC/NIV protocol for detection of Zika RNA should be 2660 used as the reference assay. 2661

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 114 | Page

Evaluations with the reference test should be conducted as per the manufacturer’s instructions 2662 for use.
2663

2664 Positive and negative samples should be subjected to both the reference test and test under 2665 evaluation. 2666 9. Sample size and sample panel composition: Sample sizes of positive and negative 2667 samples and panel composition against different values of sensitivity and specificity are provided 2668 in Tables 1 and 2. Sample sizes have been calculated assuming 95% level of significance, an 2669 absolute precision of 5%, and invalid test rate ≤5%. Appropriate sample size has to be chosen from 2670 the tables according to the values of sensitivity and specificity being claimed by the manufacturer. 2671 If a claimed sensitivity/specificity is not present in the table, the manufacturer needs to consider the 2672 sample size associated with the largest sensitivity/specificity provided in the table that is smaller to 2673 the claimed value (that is, as per the next smaller value of the sensitivity/ specificity available in 2674 the table). For example, if a manufacturer claims a sensitivity of 93%, they are required to use a 2675 sample size mentioned against 90% sensitivity.

e sensitivity/ specificity available in 2674 the table). For example, if a manufacturer claims a sensitivity of 93%, they are required to use a 2675 sample size mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 2676 usage of the sample size outlined for 85% specificity.
2677 Positive Samples:
2678  Clinical positive samples: Sample tested positive by Zika virus molecular reference assay 2679 from clinically suspect cases. 2680  Contrived positive samples: In absence of reference clinical samples, a contrived positive 2681 sample may be used.
2682 Contrived positive samples should be prepared using spiking of diluted Zika virus culture isolate 2683 in unique negative samples, as per the note below: 2684 Table 1. Sample sizes and panel composition of positive Zika virus samples for different values 2685 of sensitivity claimed by the manufacturer. 2686 Sensitivity Calculated sample size No. of Positive Samples required off] 99%# 16 20 5 between 25-30): 10
34): 5
95% 77 80 20
between 25-30): 40

claimed by the manufacturer. 2686 Sensitivity Calculated sample size No. of Positive Samples required off] 99%# 16 20 5 between 25-30): 10
34): 5
95% 77 80 20
between 25-30): 40

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 115 | Page

34): 20 90% 145 150 38
between 25-30): 74
34): 38 85% 206 210 53
between 25-30): 104
34): 53 80% 258 260 65
between 25-30): 130
34): 65 Note 1: Representative positive samples from genotype (African, Asian/American) may be included, if feasible.
Note 2: Contrived positive samples – In absence of reference clinical samples, a contrived positive sample may be used.
Contrived positive samples should be prepared using spiking of diluted Zika virus culture isolate in unique negative samples, as follows: Tissue culture fluid (3-5 different isolates with a plaque forming unit of 105-6/ml) (Heat inactivated) from reference laboratories can be obtained, spiked in serum samples (15 µl isolate + 150 µl) and can be further diluted in the ratio of 1:10, tested by the reference assay and the positive samples can be used for evaluation. The serum used for spiking isolate should be negative for Dengue virus RNA, and NS1.

#Higher sample size should be used even for assays claiming 99% sensitivity.

2687 Table 2. Sample sizes and panel composition of negative Zika virus samples for different values 2688 of specificity claimed by the manufacturer. 2689 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off]

a virus samples for different values 2688 of specificity claimed by the manufacturer. 2689 Specificity Calculated sample size No. of Negative Samples required [Sample size rounded off]

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 116 | Page

99%# 16 20 aAcute febrile cases: 10
Dengue PCR positive: 4
Chikungunya PCR positive: 1
*Japanese Encephalitis positive: 1 *West Nile Virus positive: 1
Healthy subjects from endemic regions: 3
95% 77 80 aAcute febrile cases: 40
Dengue PCR positive: 15
Chikungunya PCR positive: 5
*Japanese Encephalitis positive: 5 *West Nile Virus positive: 5
Healthy subjects from endemic regions: 10
90% 145 150 aAcute febrile cases: 76
Dengue PCR positive: 28
Chikungunya PCR positive: 9 *Japanese Encephalitis positive: 9 *West Nile Virus positive: 9
Healthy subjects from endemic regions: 19
85% 206 210 aAcute febrile cases: 105
Dengue PCR positive: 40
Chikungunya PCR positive: 13
*Japanese Encephalitis positive: 13 *West Nile Virus positive: 13
Healthy subjects from endemic regions: 26
80% 258 260 aAcute febrile cases: 130
Dengue PCR positive: 49
Chikungunya PCR positive: 16
*Japanese Encephalitis positive: 16 *West Nile Virus positive: 16
Healthy subjects from endemic regions: 33
a Acute febrile cases negative by Zika virus molecular reference assay

• Positive samples / samples spiked with culture filtrate of Japanese Encephalitis and West Nile Virus

Note:

ons: 33
a Acute febrile cases negative by Zika virus molecular reference assay

• Positive samples / samples spiked with culture filtrate of Japanese Encephalitis and West Nile Virus

Note:
If PCR positive samples for cross reactive flaviviruses are not available, commercially available RNA panels/RNA from virus isolates should be used to test cross reactivity.

#Higher sample size should be used even for assays claiming 99% specificity.

2690 10. Evaluation method: 2691 The index test and the reference tests should be run simultaneously on the sample panel to avoid 2692 false negative results by index test due to free thawing of samples or deterioration of sample quality 2693 on long term storage. 2694

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 117 | Page

11. Test reproducibility 2695 2696 2697 as follows:
    2698 2699 2700 2701 2702 samples).
    2703 2704 2705

2706 2707

2708

2709

2710 12. Acceptance criteria 2711 Sensitivity: ≥95% 2712 Specificity: ≥98% 2713 Cross reactivity with other pathogens: Nil 2714 2715

es).
2703 2704 2705

2706 2707

2708

2709

2710 12. Acceptance criteria 2711 Sensitivity: ≥95% 2712 Specificity: ≥98% 2713 Cross reactivity with other pathogens: Nil 2714 2715

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 118 | Page

2716 Agreement between sample types– Candidate tests meant for testing multiple sample matrices 2717 should demonstrate a minimum of 95% positive percent agreement (PPA) and negative percent 2718 agreement (NPA) for all specimen types. 2719

2720 14. Publication Rights: 2721 2722

2723 2724 2725 2726 2727

2728

2729 VI. References: 2730

1. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 2731 Diagnostic Assessment TGS-3.

Available at: 2732 2733 eng.pdf;sequence=1
2734

2735 2. Santiago GA, Vázquez J, Courtney S, Matías KY, Andersen LE, Colón C, Butler AE, Roulo R, 2736 Bowzard J, Villanueva JM, Muñoz-Jordan JL. Performance of the Trioplex real-time RT-PCR assay 2737 for detection of Zika, Dengue, and Chikungunya viruses. Nat Commun. 2018 Apr 11;9(1):1391. 2738 doi: 10.1038/s41467-018-03772-1. 2739

2740 3. Stone M, Bakkour S, Grebe E, Emperador DM, Escadafal C, Deng X, Dave H, Kelly-Cirino C, 2741 Lackritz E, Rojas DP, Simmons G, Rabe IB, Busch MP. Standardized evaluation of Zika nucleic acid 2742 tests used in clinical settings and blood screening. PLoS Negl Trop Dis. 2023 Mar 2743 17;17(3):e0011157.

Rojas DP, Simmons G, Rabe IB, Busch MP. Standardized evaluation of Zika nucleic acid 2742 tests used in clinical settings and blood screening. PLoS Negl Trop Dis. 2023 Mar 2743 17;17(3):e0011157. 2744

2745

2746 VII.Performance evaluation report format 2747

2748

2749

2750

2751

2752

2753

2754

2755

2756

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 119 | Page

PERFORMANCE EVALUATION REPORT FOR ZIKA REAL-TIME PCR KIT 2757 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Positive samples (provide details: clinical/spiked, strong, moderate, weak)
Negative samples (provide details clinical/spiked, including cross reactivity panel) Results 2758

Positive Negative Total Name of Zika real-time PCR kit Positive

Negative

Total

2759

Estimate (%) 95% CI Sensitivity

Specificity

2760 ● Details of cross reactivity with other flaviviruses: 2761

2762 FINAL CONCLUSION 2763 2764

ive

Total

2759

Estimate (%) 95% CI Sensitivity

Specificity

2760 ● Details of cross reactivity with other flaviviruses: 2761

2762 FINAL CONCLUSION 2763 2764

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 120 | Page

(Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 2765 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 2766 Disclaimers 2767 2768 2769 Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by …………… (supplied 2770 by ……….) 2771 2772 2773 Signature of Director/ Director-In-charge …………………… Seal………………………………. 2774 ****End of the Report 2775

2776

2777

2778

2779

2780

2781

2782

2783

2784

2785

2786

2787

2788

2789

2790

2791

2792

2793

2794

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 121 | Page

Information on Operational and Test Performance Characteristics Required from Manufacturers for 2795 Dengue/Chikungunya/ Zika IVD 2796 The manufacturer should provide the following details about the IVD: 2797

1. Instructions for Use 2798
2. Scope of the IVD: to diagnose Dengue and/or/Chikungunya and/or Zika virus 2799
3. Intended Use Statement 2800
4. Principle of the assay 2801
5. Intended testing population(cases of acute febrile illness/suspected cases of Dengue and/or 2802 Chikungunya and/or Zika virus infection) 2803

tement 2800 4. Principle of the assay 2801 5. Intended testing population(cases of acute febrile illness/suspected cases of Dengue and/or 2802 Chikungunya and/or Zika virus infection) 2803 6. Intended user(laboratory professional and/or health care worker at point-of-care) 2804 7. Detailed test protocol 2805 8. Lot/batch No. 2806 9. Date of manufacture 2807 10. Date of Expiry 2808 11. Information on operational Characteristics 2809 i. Configuration of the kit/device 2810 ii. Requirement of any additional equipment, device 2811 iii. Requirement of any additional reagents 2812 iv. Operation conditions 2813 v. Storage and stability before and after opening 2814 vi. Internal control provided or not 2815 vii. Quality control and batch testing data 2816 viii. Biosafety aspects- waste disposal requirements 2817 10. Information on Test Performance Characteristics 2818 i. Type of sample-serum/plasma/whole blood/other specimen (specify) 2819 ii. Volume of sample 2820 iii. Sample rejection criteria (if any) 2821 iv. Any additional sample processing required 2822 v. Any additional device/consumable like sample transfer device, pipette, tube, etc required 2823

iii. Sample rejection criteria (if any) 2821 iv. Any additional sample processing required 2822 v. Any additional device/consumable like sample transfer device, pipette, tube, etc required 2823

ICMR-CDSCO/IVD/GD/PROTOCOLS/02/2024 122 | Page

vi. Name of analyte to be detected 2824 vii. Pathogens targeted by the kit 2825 viii. Time taken for testing 2826 ix. Time for result reading and interpretation 2827 x. Manual or automated(equipment)reading 2828 xi. Limit of detection 2829 xii. Diagnostic sensitivity 2830 xiii. Diagnostic specificity 2831 xiv. Stability and reproducibility (including data) 2832 xv. Training required for testing (if any) 2833 xvi. If yes, duration 2834 xvii. Details of Cut-off and /or Equivocal Zone for interpretation of test 2835 xviii. Details of cross reactivity, if any 2836 xix. Interpretation of invalid and indeterminate results to be provided 2837 xx. It is recommended to provide data demonstrating the precision 2838

2839 *Please mention “Not applicable” against sections not pertaining to the kit. 2840

2841

2842 ****End of the Document 2843

2844