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54 Inviting comments on standard IVD evaluation protocol drafted by ICMR and CDSCO 3 2025-Aug-12 4130 KB

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ICMR-CDSCO/IVD/GD/PROTOCOLS/11/2025 Page1of2

Indian Council of Medical Research and Central Drugs Standard Control Organization Department of Health Research and Drugs Controller General of India Ministry of Health and Family Welfare Government of India Document No.: ICMR-CDSCO/IVD/GD/PROTOCOLS/11/2025

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:

PerformanceevaluationprotocolforInfluenzavirusmoleculardetectionand/or differentiation assay (single plex/multiplex format)
PerformanceevaluationprotocolforSARS-CoV-2moleculardetectionassay(single plex/multiplex format)
Performance evaluation protocol for Respiratory Syncytial Virus molecular detection assay (single plex/multiplex format)
Performance evaluation protocol for Influenza virus and SARS-CoV-2 molecular detection differentiation assay (multiplex format)
PerformanceevaluationprotocolforInfluenzavirus,SARS-CoV-2andRSVmoleculardetection and differentiation assay (multiplex format)
Performance evaluation protocol for Malaria rapid diagnostic test (RDT) for P falciparum and/or P vivax
Performance evaluation protocol for Malaria ELISA assay
Performance evaluation protocol for Malaria real time PCR assay
Field evaluation protocol for combo Malaria Rapid Diagnostic Test (RDT) kits (detecting P vivax and P falciparum)
Performance evaluation protocol for Nipah virus Real Time PCR
Performance evaluation protocol for Chandipura virus Real Time PCR
Performanceevaluationprotocolformultiplexrespiratoryvirus(expandedpanel)Real Time PCR
Performance evaluation protocol for Dengue IgG RDT
Performance evaluation protocol for Dengue IgM/IgG Combo RDT
Performance evaluation protocol for Dengue IgG ELISA

These protocols are now being placed in the public domain for comments from relevant stakeholders. This window of opportunity will close on 25th August 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 25th August 2025, at ivdevaluation@gmail.com as per the enclosed format. Once the public

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Consultation period concludes, all comments will be reviewed and considered in finalizing the draft protocols before final clearance by ICMR and CDSCO.

Dated: 11thAugust2025 Place: New Delhi

STANDARDIVDPERFORMANCEEVALUATIONPROTOCOL STAKEHOLDERFEEDBACKFORM

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

Name:

Designation and Affiliation:

1 STANDARD PERFORMANCE 2 EVALUATION PROTOCOLS 3

4 DRAFT FOR STAKEHOLDER COMMENTS 5 Influenza virus, SARS-CoV-2, Respiratory Syncytial Virus
6 In-Vitro Diagnostics 7

8 ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 9

12 AUGUST, 2025 New Delhi, India

Influenza, SARS-CoV-2, RSV Molecular IVD Performance Evaluation Protocols ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 1 of 41

Table of Contents 13 S.N. Topic Page Number 1.
General Guidelines 2 2.
Protocol A: Protocol for performance evaluation of influenza virus targets in single plex or multiplex molecular assay formats 6 3.
Protocol B: Protocol for performance evaluation of SARS-CoV-2 in single plex or multiplex molecular assay formats 14 4.
Protocol C: Protocol for performance evaluation of Respiratory Syncytial Virus (RSV) in single plex or multiplex molecular assay formats 21 5.
Protocol D: Protocol for performance evaluation of influenza viruses and SARS-CoV-2 in multiplex molecular assay format 28 6.
Protocol E: Protocol for performance evaluation of influenza viruses, SARS-CoV-2 and RSV in multiplex molecular assay format 31 7.
Performance Evaluation Report Format 34 8.
Annexure-1: Information on operational and test performance characteristics required from manufacturers 39

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GENERAL GUIDELINES 37

38 Protocols for performance evaluation of in vitro molecular diagnostic kits for detection and 39 differentiation of Influenza virus and/or SARS-CoV-2 and/or RSV
40

Introduction: 41 This document provides a framework for evaluating the performance characteristics of in vitro 42 diagnostic (IVD) kits used in identifying and distinguishing various strains of Influenza viruses 43 and/or SARS-CoV-2 and/or RSV, aligning with international standards to ensure reliability and 44 accuracy in diagnosis. The coronavirus diseases 2019 (COVID-19) pandemic, caused by the 45 SARS-CoV-2 virus, has necessitated the rapid development and validation of in vitro molecular 46 diagnostic kits. These kits are crucial for the timely detection and differentiation of major 47 respiratory viruses (influenza/SARS-CoV-2/RSV) to control their spread. This protocol outlines a 48 systematic approach for validating these diagnostic kits to ensure their accuracy, sensitivity, 49 specificity, and reliability. 50 Although SARS-CoV -2 is no longer a public health emergency globally, it is prudent to 51 implement integrated surveillance for Influenza, SARS-CoV-2 and other respiratory viruses, 52 making differential diagnosis for these viruses essential. Additionally, timely diagnosis of other 53 respiratory viruses, particularly Respiratory syncytial virus (RSV), is crucial for providing 54 effective clinical management to pediatric cases.
55 This document provides guidance for single plex or multiplex assays for the differential diagnosis 56 of Influenza and/or SARS-CoV-2 and/or RSV. It outlines the evaluation of IVD devices/kits 57 intended for the detection and differentiation of influenza virus strains and/or detection of SARS- 58 CoV-2 and/or detection and differentiation of RSV using nucleic acid detection methods as 59 outlined in the scope below. This includes IVD devices/kits that detect and differentiate between 60 influenza virus types (Influenza A or B), subtypes (A (H1N1) pdm09 or A (H3N2)), and/or 61 multiple influenza virus types/subtypes; kits that identify only SARS-CoV-2, as well as kits that 62 only detect and/or differentiate RSV. Additionally, this protocol may be used for multiplex IVD 63 devices/kits designed to simultaneously detect Influenza A & B (with or without subtyping), and/or 64 SARS-CoV-2, and/or RSV. This document outlines the following aspects of performance 65 evaluation of IVD devices/kits as per the scope outlined in the document: 66 1.1 The procedure for validating entities to determine operational parameters of IVD 67 devices/kits that detect influenza virus gene segment(s). 68 1.2 T procedure for validating entities to determine operational parameters of IVD devices/kits 69 that detect SARS-CoV-2 gene segment(s).
70 1.3 The procedure for validating entities to determine operational parameters of IVD 71 devices/kits that detect RSV gene segment(s).
72 1.4 The techniques for identifying influenza virus/SARS-CoV-2/RSV nucleic acid targets in 73 single-plex or multiplex formats (using appropriate protocols listed in the document).
74

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1.5 This document is not useful for performance evaluation of serological assays for detection 75 of antigen and antibody for influenza viruses/SARS-CoV-2/RSV. The IVD device/kit to be 76 validated is henceforth known as the “Kit under Evaluation.” 77 2. Objective:
78 This document aims to offer a comprehensive set of instructions for evaluating the performance of 79 molecular IVD assays mentioned in the scope below for detecting Influenza A and Influenza B 80 viruses with/without subtyping, and other common respiratory viruses such as SARS-CoV-2 and 81 RSV. This evaluation will focus on measuring the analytical sensitivity and specificity, cross- 82 reactivity, repeatability, and reproducibility as compared against a reference assay using clinical 83 sample panel. 84 In brief, the objectives are as follows: 85 2.1 To validate the performance characteristics of in vitro molecular diagnostic kits for 86 detecting Influenza A & B (with/without subtyping)/ SARS-CoV-2/ RSV. 87 2.2 To ensure the kits under evaluation meet the necessary standards for sensitivity, 88 specificity, repeatability, and reproducibility. 89 2.3 To evaluate the cross-reactivity of the kits with other respiratory viruses. 90 3. Scope: 91 This guideline is solely for the evaluation and establishment of the performance characteristics of 92 IVD kits and devices designed for the detection and subtyping of commonly circulating seasonal 93 Influenza viruses (Influenza A(H1N1) pdm09, Influenza A(H3N2), Influenza B(Yamagata) and 94 Influenza B(Victoria) subtypes) and/or other common respiratory viruses such as SARS-CoV-2 95 and RSV, using single or multiplex molecular assays (as outlined in the scope below) intended for 96 human clinical samples. This document is a guide to assess: 97 3.1 The analytical assay performance characteristics with clinical specimens for the detection 98 and/or differentiation of influenza viruses. (Protocol A)
99 3.2 The analytical assay performance characteristics with clinical specimens for the detection 100 of SARS-CoV-2 (Protocol B)
101 3.3 The analytical assay performance characteristics with clinical specimens for the detection 102 of RSV (Protocol C)
103 3.4 The analytical performance characteristics of multiplex assay for detection of two or more 104 of these viruses by combining Protocols A, B & C as per the kit format.
105 3.5 Analytical performance characteristics which should include sensitivity, specificity, cross- 106 reactivity, and lot-to-lot variation including functionality of devices that identify and/or 107 differentiate influenza viruses, SARS-CoV-2 and/or RSV depending on the kit format. 108 3.6 The performance of the kit, only if the kit includes an internal control (preferably 109 endogenous, or exogenous).
110

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3.7 This document may also apply to forthcoming influenza, SARS-CoV-2 and RSV molecular 111 diagnostic devices that do not fit within these current classifications.
112 3.8 The document will serve as a reference for assessing kits based on Nucleic Acid 113 Amplification Test (single plex or multiplex assays) as listed below: 114

115 3.8.1 Real-time Reverse Transcription Polymerase Chain Reaction format (rRT-PCR): 116 including Real-time PCR probe-based assays or non-probe based assays 117 3.8.2 Other NAT testing platforms such as LAMP/RPA, and other closed system platforms 118 such as TrueNat /cartridge-based assays 119

120 Note: This protocol is not suitable for the kits where amplicons are handled outside the 121 amplification system. 122 4. Requirements:
123 4.1 Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 124 the kit to be evaluated works in a closed system format, the manufacturer needs to supply 125 the required equipment and consumables. 126 4.2 Evaluation sites/laboratories (With required equipment) 127 4.3 Reference test kits 128 4.4 Characterized samples for evaluation panel 129 4.5 Laboratory supplies
130

131 5. Ethical approvals:
132 Laboratory validation of IVDs using irreversibly de-identified samples is exempted from ethics 133 approval as per ICMR’s Guidance on Ethical Requirements for Laboratory Validation Testing, 134 2024. A self-declaration form as provided in ICMR guidelines to be submitted by the 135 investigators to the institutional authorities and ethics committee for information 136 (https://ethics.ncdirindia.org/asset/pdf/Guidance_on_Ethical_Requirements_for_Laboratory_ 137 Validation_Testing.pdf ) 138 6. Procedure: 139 6.1 Study design/type: Diagnostic accuracy study using leftover irreversibly de-identified 140 archived clinical samples.
141 6.2 Evaluation site/laboratory considerations: Identified IVD kit evaluation laboratories should 142 establish their proficiency through
143 6.2.1 Accreditation for at least one of the Quality management systems (accreditation for 144 Testing Lab / Calibration Lab (ISO: 17025), Medical Lab (ISO 15189), PT provider (ISO: 145 17043) or CDSCO approved Reference laboratory. 146

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6.2.2 Have sufficient numbers of archived as well as contemporary clinical specimens positive 147 for respiratory viruses targeted by the kit under evaluation (Influenza A(H1N1)pdm09, 148 A(H3N2), B(Yamagata), B(Victoria), and/or SARS-CoV-2 and/or RSV A & B), with aliquots 149 stored at -80 ℃ deep freezers or in lyophilized form. 150 6.2.3 Virus strains should be well-characterized by ICMR approved or US FDA/ ATAGI 151 Australia/PMDA Japan approved/WHO Pre-Qualified reference assay and/or by influenza 152 virus HA gene/segment or gene-specific sequencing (for SARS-CoV-2 and RSV) or Next- 153 Generation Sequencing. 154 6.2.4 Have a minimum BSL-2 level facility with trained manpower and at least two different 155 Real Time platforms to perform molecular diagnostic assays for Influenza virus and other 156 respiratory viruses. 157 6.2.5 Have a good record of External Quality Assurance programs for influenza, SARS-CoV- 158 2, and other respiratory viruses. 159 6.2.6 Staff training: All the staff involved in IVD kit evaluation should undergo hands-on 160 training and competency testing on the following: 161 6.2.6.1 Preparation & characterization of kit evaluation panel
162 6.2.6.2 Handling of respiratory virus PCR kits received for performance evaluation 163 (Verification/Storage/Unpacking etc). 164 6.2.6.3 Testing, interpreting, recording of results & reporting 165 6.2.6.4 Data handling, data safety & confidentiality 166

167 6.3 Performance characteristics: To be assessed for all assay targets of influenza A/B, 168 SARS-CoV-2 and RSV (single plex or multi-plex assays) 169 6.3.1 Analytical Sensitivity and specificity
170 6.3.2 Cross-reactivity
171 6.4.3 Repeatability 172 6.4.4 Reproducibility 173

174

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176

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Protocol A
180 Evaluation of performance characteristics of Molecular Kit detecting influenza A & B 181 viruses, and subtyping into A (H1N1) pdm 09, A(H3N2), B(Yamagata) & B(Victoria) in 182 single plex or multiplex format 183

Objective:
184 1.1 To evaluate the performance of molecular IVD device /KIT for detection and 185 differentiation of Influenza viruses as per the scope outlined in this document.
186 1.2 To ensure the kits under evaluation meet the necessary standards for sensitivity, 187 specificity, repeatability, and reproducibility.
188 1.3 To evaluate the cross-reactivity of the kits with other respiratory viruses. 189

190 2. Evaluation of performance characteristics should be done for the following 191 parameters: 192 2.1 Sensitivity and specificity 193 2.2 Cross-reactivity 194 2.3 Repeatability 195 2.4 Reproducibility 196

197 3. Panel development: Clinical sample (archived/contemporary) panel for testing: 198 3.1 Contemporary leftover irreversibly de-identified clinical/archived respiratory samples 199 (in VTM) for the panel should be irreversibly de-identified. 200 3.2 Samples to be used for panel preparation shall be stored properly at – 80 ℃ or 201 lyophilized. 202 3.3 Unless the manufacturer has specific requirement of nucleic acid extraction kit, the 203 validation laboratory can use WHO Pre-Qualified/ US FDA/ ATAGI Australia/ PMDA 204 Japan approved/ICMR validated total RNA / viral RNA extraction kits for the evaluation.
205 3.4 Clinical samples for evaluation should be characterized by a reference kit / 206 Sequencing/NGS. 207 3.5 All positive samples should be confirmed positive for the target pathogens by the 208 reference assay. 209 3.6 All negative samples should be confirmed negative for the target pathogens by the 210 reference assay. 211

212

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Sample size and sample panel composition for evaluation of performance 214 characteristics: 215 Sample sizes of positive and negative samples of the analyte/pathogen targeted by the kit 216 against different values of sensitivity and specificity are provided in Table 1. Sample sizes 217 have been calculated assuming 95% level of significance, an absolute precision of 5%, and 218 invalid test rate ≤5%. Appropriate sample size has to be chosen from the tables according to 219 the values of sensitivity and specificity being claimed by the manufacturer. If a claimed 220 sensitivity/specificity is not present in the table, the manufacturer needs to consider the sample 221 size associated with the largest sensitivity/specificity provided in the table that is smaller to the 222 claimed value (that is, as per the next smaller value of the sensitivity/ specificity available in 223 the table). For example, if a manufacturer claims a sensitivity of 93%, they are required to use 224 a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would 225 require usage of the sample size outlined for 85% specificity. Sample sizes are calculated using 226 the formulae: 227

228 𝑛𝑠𝑒 ≥𝑍2 x 𝑆𝑒 (1 −𝑆𝑒) 𝑑2 x (1 −IR)
229 𝑛𝑠𝑝 ≥𝑍2 x 𝑆𝑝 (1 −𝑆𝑝) 𝑑2 x (1 −IR)
230

231

232 · n (se) is the number of positive samples. 233 · n (sp) is the number of negative samples. 234 · Z2 is the critical value from the standard normal distribution corresponding 235 to the desired confidence level (95% CI corresponds to Z2 =1.96). 236 · Se is the predetermined sensitivity. 237 · Sp is the predetermined specificity. 238 · d is the predetermined marginal error (5%) 239 · IR is the invalid test rate 240 Sample sizes for positive samples and their composition for evaluating subtyping are 241 provided in Table 2. 242

243 Table 1. Sample sizes per target pathogen for different values of sensitivity/ specificity 244 claimed by the manufacturer. 245

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Sensitivity/ Specificity Sample size: Minimum number of positive samples¥ Composition of positive samples# Sample size: Minimum number of negative samples (rounded) ¥ Minimum number of cross reactive* samples among the negative samples 99% 16 (rounded to 20 for better distribution of samples) Strong positive = 06 Moderate positive = 07 Weak positive = 07 20 5 95% 77 (rounded to 80 for better Strong positive = 24 Moderate positive = 28 Weak positive = 28 80 20 90% 146 (rounded to 155 for better Strong positive = 45 Moderate positive = 55 Weak positive =55 150 38 85% 207 (rounded to 215 for better Strong positive = 63 Moderate positive = 76 Weak positive = 76 210 53 80% 259 (rounded to 260 for better Strong positive = 78 Moderate positive = 91 Weak positive = 91 260 65 #Strong positive: (Ct value <25) Moderate positive: (Ct value between 25-30) Weak positive: (Ct value >30 and and ≤ 34)

¥ Equal distribution of positive nasopharyngeal and/or oropharyngeal swabs in virus transport medium (VTM) to be used

Samples positive for common respiratory viruses (such as SARS-CoV-2, Parainfluenza viruses, Adenoviruses, Rhinoviruses, Respiratory Syncytial Virus (including its types and subtypes), common human coronaviruses), other than the ones targeted by the kit under evaluation. Equal distribution of cross-reactive viruses is desirable. It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 246 and specificity; however, a higher sample size is suggested to ensure adequate power of 247 the study in case the kit falls short of claimed performance characteristics. 248 Table 2. Sample sizes for positive samples and their composition for evaluating subtyping
249 Sample size* (per target pathogen) Minimum total number of positive samples (rounded figures) Influenza A (H1N1) pdm09 Influenza A/H3N2 Influenza B Sensitivity Minimum number of nasopharyngeal swabs/ oropharyngeal swabs (rounded figures) Minimum number of nasopharyngeal swabs/ oropharyngeal swabs (rounded figures) Minimum number of nasopharyngeal swabs/ oropharyngeal swabs (rounded figures) 99% 20 20 20 60 95% 80 80 80 240

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90% 150 150 150 450 85% 210 210 210 630 80% 260 260 260 780 *Combination of strong, moderate and weak positive samples should be considered as per the information provided in Table 1. It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 250 251 252 4.1 Repeatability testing will be performed on 3 positive (strong, moderate and weak 253 positive) and 3 Negative samples (within the selected positive and negative samples) per 254 target pathogen 5 times (replicates of 5).
255

256 5. Methodology: 257 5.1 Samples should be tested in parallel with the Kit Under Evaluation and the reference 258 assay. The ICMR-NIV RT-qPCR assay for Influenza/SARS-CoV-2 or WHO Pre- 259 Qualified/ US FDA/ ATAGI Australia/ PMDA Japan approved kit will be considered as 260 the reference assay for these parameters. 261 5.2 The validation laboratory can use the established total RNA / viral RNA extraction 262 protocol for the evaluation.
263 5.3 The instruction for the assay setup and the interpretation of the results will be as per 264 the protocol outlined by the manufacturer of the reference test and the kit under evaluation.
265 5.4 The results shall be compared with the reference assay for sensitivity and specificity 266 calculations. 267 5.5 If there is a discrepancy observed in the results with the index test, this discrepancy 268 should be taken as discordant. Repetition of the assay may introduce bias. If the reference 269 kit itself has failed, then these samples with discrepancies should be discarded, and new 270 well-characterized samples should be used instead.
271 True positive samples: These are samples positive by both reference assay and index test. 272 True negative samples: These are samples negative by both reference assay and index test. 273 False positive samples: These are samples negative by reference assay and positive by 274 index test. 275 False negative samples: These are samples positive by reference assay and negative by 276 index test. 277 5.6 The interpretation for internal control (preferably endogenous, or exogenous) will be 278 as per manufacturer's instruction.
279 5.7 PCR should be performed using IVD-approved machines. If any equipment(s) is 280 specified in the IFU of the index test, it should be used for the evaluation, and it should be 281 provided by the manufacturer if not available within the lab’s IVD evaluation scope.
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Real-time closed systems/devices awaiting evaluation should be provided by the 283 manufacturer along with all necessary components, supplies and reagents. 284 The details on the Real-time Equipment used for validation should be recorded, including 285 calibration status. 286

287 6. Cross-reactivity Analysis: 288 6.1 Objective:
289 To assess the primer-probe set for true detection of influenza viruses and assess its cross- 290 reactivity with other respiratory viruses. 291 6.2 Methodology: 292 6.2.1 Potential cross-reactivity of the kit shall be ruled out by testing other 293 respiratory pathogen positive samples (N=30), with equal representation (n=5 each) 294 of samples positive for SARS-CoV-2, Parainfluenza viruses, Adenoviruses, 295 Rhinoviruses, Respiratory Syncytial Virus, common human coronaviruses).* 296 6.2.2 Cross-reactivity will be assessed by comparing the results of these samples 297 using kit under evaluation and reference kit. 298 6.2.3 The kit targets should not show any amplification with other respiratory 299 viruses (ORVs). If amplification is observed for ORV then the kit will fail 300 validation and the same needs to be mentioned in the report.
301

302

For multiplex assays targeting influenza, SARS-CoV-2, and RSV, samples positive for 303 these viruses may be suitably interchanged for assessing cross-reactivity, apart from the 304 ORV panel. (i.e. Influenza A positive samples may be used for detecting cross-reactivity 305 against Influenza B) 306

307 7. Acceptance criteria for the kit:
308 Sensitivity for each pathogen/ type/ subtype: ≥95%
309 Specificity for each pathogen/ type/ subtype: ≥99%
310 Cross-reactivity: Nil 311 Invalid test rate: ≤5% 312 To achieve at least the performance characteristics outlined in the acceptance criteria, ≥80 313 positive samples and ≥20 negative samples should be tested for evaluation for each 314 pathogen/ type/ subtype. 315 8. Repeatability Assessment:
316

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8.1 Objectives:
317 To assess the repeatability of the detection of Influenza virus and its subtypes using the 318 kit under evaluation
319 8.2 Sample size:
320 3 positive samples (strong, moderate and weak positive-as per the Ct values outlined in 321 the document) and 3 negative samples for each target pathogen should be tested 5 times. 322

323 8.3 Result: Concordance should be 100% based on positive and negative test result 324 (qualitative). 325

326 9. Precision (Reproducibility): 327 Lot to Lot Reproducibility 328 9.1 Objectives: To assess Precision (Reproducibility) among 3 different lots of the 329 kit under evaluation. 330 9.2 Sample size: Three lots of an assay shall be evaluated. Sample size for lot-to-lot 331 reproducibility should be as follows:
332 • First lot of the assay: should be tested on statistically significant number of positive 333 and negative samples as calculated in the protocol.
334 • Second lot of the assay: should be tested on 25 samples (15 positive samples 335 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 336 samples).
337 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 338 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 339

340 9.3 Result: Concordance should be 100% based on positive and negative test result 341 (qualitative) 342 10. Internal Control Analysis: 343 10.1 Monitor the internal control (preferably RNaseP or other housekeeping gene) to 344 ensure consistent extraction and amplification efficiency across samples and runs. 345 10.2 Ct-values of internal controls should be within the manufacturer’s prescribed limit. 346 10.3Tests will be marked invalid if Ct-values are outside the prescribed limit.
347

348 11. Blinding of Laboratory Staff: 349 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should 350 be blinded to the status of the clinical samples. The PI of the evaluation exercise should 351 remain unblinded, i.e., privy to the status of the samples. Another senior laboratory staff 352

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selected by the PI may remain unblinded and carry out coding of samples and dispensing 353 them into similar-looking vials to be used for testing, and maintaining the database of 354 results. Staff performing the reference test and the test under evaluation, interpretation of 355 the test result, and entering the results against the coded samples in the database, should 356 remain blinded to the status of samples till the completion of evaluation. The data should 357 be analyzed only by the PI of the evaluating lab. Refer to Fig. 1. 358

359 Fig.1: Blinding in evaluation exercise 360

361

362

363 12. Publication Rights:
364 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead 365 author(s). 366 13. Conclusion: 367 Based on the comprehensive evaluation conducted, the [Kit & Manufacturer’s Name] Influenza 368 Virus RT-PCR Assay has been found [Satisfactory/Not Satisfactory] for its intended in vitro 369 diagnostic (IVD) use.
370

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The assay demonstrates [Strengths/Concerns] in terms of sensitivity, specificity, and 371 performance characteristics compared to established reference IVD approved RT-PCR kits. 372

373 After following due procedure as defined in this document, once any kit is found to be Not 374 of Standard Quality, thereafter, no request for repeat testing of the same kit will be 375 acceptable.
376 Any request of re-validation from the same manufacturer for the same test type will only be 377 entertained after a minimum of 3 months and only if a high-level technical summary of 378 modifications or functional improvements to the kit design is submitted, without explicit 379 disclosure of proprietary information. 380 Clinical samples are precious, therefore, repeat evaluation of a kit using the same/ different 381 well-characterized sample panel at a different laboratory may be considered only for kits 382 which claim high performance characteristics (sensitivity and specificity 95% and above), 383 but which fail the performance evaluation by a margin of 5%. 384

385 14. Performance evaluation report format 386 The performance evaluation report format (page 34) is designed for multiplex assays with 387 several targets. It should be modified and used accordingly for single plex assays/multiplex 388 assays with fewer targets. 389

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Protocol B 404 Evaluation of performance characteristics of Molecular Kit detecting SARS-CoV-2 in 405 single plex or multiplex format 406

Objective: 407 1.1. To validate the performance characteristics of in vitro molecular diagnostic kits for 408 detecting SARS-CoV-2 as per the scope outlined in this document. 409 1.2. To ensure the kits under evaluation meet the necessary standards for sensitivity, 410 specificity, repeatability, and reproducibility. 411 1.3. To evaluate the cross-reactivity of the kits with other respiratory viruses. 412

413 2. Evaluation of Performance characteristic should be done for the following:
414 2.1 Sensitivity and specificity 415 2.2 Cross-reactivity
416 2.3 Repeatability 417 2.4 Reproducibility 418 3. Panel development: Clinical sample (archived/ contemporary) panel for testing: 419 3.1 Contemporary leftover irreversibly de-identified clinical/archived respiratory samples 420 in VTM for the panel should be irreversibly de-identified. 421 3.2 Samples to be used for panel preparation shall be stored properly at – 80 ℃ or 422 lyophilized. 423 3.3 Unless the manufacturer has specific requirement of nucleic acid extraction kit, the 424 MDTLs/ validation laboratory can use WHO Pre-Qualified/ US FDA/ ATAGI Australia/ 425 PMDA Japan approved/ICMR validated an established total RNA / viral RNA extraction 426 kits for the evaluation.
427 3.4 Clinical samples for evaluation should be characterized by a reference kit / 428 Sequencing/NGS. 429 3.5 All positive samples should be confirmed positive for the target pathogens by the 430 reference assay. 431 3.6 All negative samples should be confirmed negative for the target pathogens by the 432 reference assay. 433

434 4. Sample size and sample panel composition for evaluation of performance characteristics:
435

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Sample sizes of positive and negative samples of SARS-CoV-2 against different values of 436 sensitivity and specificity are provided in Table 3. Sample sizes have been calculated assuming 437 95% level of significance, an absolute precision of 5%, and invalid test rate ≤5%. Appropriate 438 sample size has to be chosen from the tables according to the values of sensitivity and 439 specificity being claimed by the manufacturer. If a claimed sensitivity/specificity is not present 440 in the table, the manufacturer needs to consider the sample size associated with the largest 441 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as per 442 the next smaller value of the sensitivity/ specificity available in the table). For example, if a 443 manufacturer claims a sensitivity of 93%, they are required to use a sample size mentioned 444 against 90% sensitivity. Similarly, a claim of 87% specificity would require usage of the 445 sample size that is outlined for 85% specificity. Sample sizes are calculated using the formulae: 446

447 𝑑2 x (1 −IR)
448 𝑑2 x (1 −IR)
449

450

451 · n (se) is the number of positive samples. 452 · n (sp) is the number of negative samples. 453 454 455 456 457 458 459

460

461 Table 3. Sample sizes for different values of sensitivity/ specificity claimed by the manufacturer. 462 Sensitivity/ Specificity Sample size: Minimum number of positive samples¥ Composition of positive samples# Sample size: Minimum number of negative samples (rounded)¥ Minimum number of cross reactive* samples among the negative samples

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99% 16 (rounded to 20 for better Weak positive = 07 20 5 95% 77 (rounded to 80 for better Weak positive = 28 80 20 90% 146 (rounded to 155 for better distribution of samples) Strong positive = 45 Moderate positive = 55 Weak positive =55 150 38 85% 207 (rounded to 215 for samples) Strong positive = 63 Moderate positive = 76 Weak positive = 76 210 53 80% 259 (rounded to 260 for samples) Strong positive = 78 Moderate positive = 91 Weak positive = 91 260 65 #Strong positive: (Ct value <25) Moderate positive: (Ct value between 25-30) Weak positive: (Ct value >30 and ≤ 34)

¥ Nasopharyngeal/ oropharyngeal swabs in virus transport medium (VTM) to be used *Samples positive for common respiratory viruses (such as Influenza (including its types and subtypes), Parainfluenza viruses, Adenoviruses, Rhinoviruses, Respiratory Syncytial Virus (including its types and subtypes), common human coronaviruses), other than the ones targeted by the kit under evaluation. Equal distribution of cross-reactive viruses is desirable. It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 463 464 465 4.1 Repeatability testing will be performed on 3 positive (strong, moderate and weak 466 positive) and 3 negative samples (within the selected positive and negative samples) per 467 target pathogen 5 times (replicates of 5).
468

469 5. Methodology: 470 5.1 Samples should be tested in parallel with the Kit Under Evaluation and the reference 471 assay. The ICMR-NIV RT-qPCR assay for Influenza/SARS-CoV-2 or WHO Pre- 472 Qualified/ US FDA/ PMDA Japan/ ATAGI Australia approved kit will be considered 473 as the reference assay for these parameters. 474 5.2 The validation laboratory can use established total RNA / viral RNA extraction 475 protocol for the evaluation.
476 5.3 The instruction for the assay setup and the interpretation of the results will be as per 477 the protocol outlined by the manufacturer of the reference test and the kit under 478 evaluation. The results shall be compared with the reference assay for sensitivity and 479 specificity calculations. 480 5.4 If there is a discrepancy observed in the results with the index test, this discrepancy 481 should be taken as discordant. Repetition of the assay may introduce bias. If the 482 reference kit itself has failed, then these samples with discrepancies should be 483 discarded, and new well-characterized samples should be used instead.
484 True positive samples: These are samples positive by both reference assay and index test. 485

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 17 of 41

True negative samples: These are samples negative by both reference assay and index test. 486 487 index test. 488 489 index test. 490

491 5.5 The interpretation for internal control (preferably endogenous, or exogenous) will be 492 as per manufacturer's instruction.
493 5.6 PCR should be performed using IVD-approved machines. If any equipment(s) is 494 specified in the IFU of the index test, it should be used for the evaluation, and it should 495 be provided by the manufacturer if not available within the lab’s IVD evaluation 496 scope.
497 Real-time closed systems/devices awaiting evaluation should be provided by the 498 manufacturer along with all necessary components, supplies and reagents. 499 The details on the Real-time Equipment used for validation should be recorded, 500 including calibration status. 501 The details on the Real-time Equipment used for validation should be recorded 502 including calibration status. 503

504 6. Cross-reactivity Analysis: 505 6.1 Objective:
506 To assess the primer-probe set for true detection of SARS-CoV-2 and assess its cross- 507 reactivity with other respiratory viruses. 508 6.2 Methodology: 509 6.1.1 Potential cross-reactivity of the kit shall be ruled out by testing other respiratory 510 pathogen positive samples (N=30), with equal representation (n=5 each) of 511 samples positive for Influenza, Parainfluenza viruses, Adenoviruses, 512 Rhinoviruses, Respiratory Syncytial Virus, common human coronaviruses).*
513 6.1.2 Cross-reactivity will be assessed by comparing the results of these samples using 514 kit under evaluation and reference kit. 515 6.1.3 The kit targets should not show any amplification with other respiratory viruses 516 (ORVs). If amplification is observed for ORV then the kit will fail validation 517 and the same needs to be mentioned in the report.
518

519 7. Acceptance criteria for the kit:
520

521 Sensitivity: ≥95%
522 Specificity: ≥99%

523 524

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 18 of 41

525 To achieve at least the performance characteristics outlined in the acceptance criteria, ≥80 526 positive samples and ≥20 negative samples should be tested for evaluation for each 527 pathogen/ type/ subtype. 528 8. Repeatability Assessment:
529 8.1 Objectives: To assess the repeatability of the detection of SARS-CoV-2 using the kit 530 under evaluation
531

532 8.2 Sample size: Five replicates of 3 positive samples (strong, moderate and weak positive- 533 as per the Ct values outlined in the document), and five replicates of 3 negative samples 534 for SARS-CoV-2 should be tested. For multiplex panels, these sample numbers shall 535 be used per target pathogen for repeatability assessment. 536

537 8.3 Result: Concordance should be 100% based on positive and negative test result 538 (qualitative). 539

540

541 9. Precision (Reproducibility): 542 Lot to Lot Reproducibility 543

544 9.1 Objectives: To assess precision (reproducibility) among 3 different lots of the kit 545 under evaluation.
546 9.2 Sample size: Lot to lot variation testing: Three lots of an assay shall be evaluated. 547 Sample size for lot-to-lot reproducibility should be as follows:
548 549 and negative samples as calculated in the protocol.
550 • Second lot of the assay: should be tested on 25 samples (15 positive samples 551 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 552 samples).
553 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 554 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 555

556 9.3 Result: Concordance should be 100% based on positive and negative test result 557 (qualitative). 558

559 10. Internal Control Analysis: 560 10.1 Monitor the internal control (preferably RNaseP or other housekeeping gene) to 561 ensure consistent extraction and amplification efficiency across samples and runs. 562

563

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 19 of 41

10.2 Ct-values of internal controls should be within the manufacturer’s prescribed limit. 564

565 10.3 Tests will be marked invalid if Ct-values are outside the prescribed limit.
566

567 11. Blinding of Laboratory Staff: 568 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should 569 be blinded to the status of the clinical samples. The PI of the evaluation exercise should 570 remain unblinded, i.e., privy to the status of the samples. Another senior laboratory staff 571 selected by the PI may remain unblinded and carry out coding of samples and dispensing 572 them into similar-looking vials to be used for testing, and maintaining the database of 573 results. Staff performing the reference test and the test under evaluation, interpretation of 574 the test result, and entering the results against the coded samples in the database, should 575 remain blinded to the status of samples till the completion of evaluation. The data should 576 be analyzed only by the PI of the evaluating lab. Refer to Fig. 1 in Protocol A. 577

578 12. Conclusion: 579 Based on the comprehensive evaluation conducted, the [Kit & Manufacturer’s Name] SARS-CoV- 580 2 RT-PCR Assay has been found [Satisfactory/Not Satisfactory] for its intended in vitro 581 diagnostic (IVD) use.
582 The assay demonstrates [Strengths/Concerns] in terms of sensitivity, specificity, and performance 583 characteristics compared to established reference IVD approved RT-PCR kits. 584

585 13. Publication Rights: 586 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead author(s). 587

588 589 590 acceptable.
591 592 593 594 595 596 597 598 599

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600 14. Performance evaluation report format: 601 The performance evaluation report format (page 34) is designed for multiplex assays with several 602 targets. It should be modified and used accordingly for single plex assays/multiplex assays with 603 fewer targets. 604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 21 of 41

Protocol C 629 Evaluation of performance characteristics of Molecular Kit detecting Respiratory 630 Syncytial Virus (RSV) in single plex or multiplex format 631

Objective: 632 1.1. To validate the performance characteristics of in vitro molecular diagnostic kits for 633 detecting and/or differentiating RSV A/B as per the scope outlined in this document. 634 1.2. To ensure the kits under evaluation meet the necessary standards for sensitivity, 635 specificity, repeatability, and reproducibility. 636 1.3. To evaluate the cross-reactivity of the kits with other respiratory viruses. 637

638 2. Evaluation of Performance characteristic should be done for the following:
639 2.1 Sensitivity and specificity 640 2.2 Cross-reactivity
641 2.3 Repeatability 642 2.4 Reproducibility 643 3. Panel development: Clinical sample (archived/ contemporary) panel for testing: 644 3.1 Contemporary leftover irreversibly de-identified clinical/archived respiratory samples 645 in VTM for the panel should be irreversibly de-identified. 646 3.2 Samples to be used for panel preparation shall be stored properly at – 80 ℃ or 647 lyophilized. 648 3.3 Unless the manufacturer has specific requirement of nucleic acid extraction kit, the 649 MDTLs/ validation laboratory can use WHO Pre-Qualified/ US FDA/ ATAGI Australia/ 650 PMDA Japan approved/ ICMR validated an established total RNA / viral RNA extraction 651 kits for the evaluation.
652 3.4 Clinical samples for evaluation should be characterized by a reference kit / 653 Sequencing/NGS. 654 3.5 All positive samples should be confirmed positive for the target pathogens by the 655 reference assay. 656 3.6 All negative samples should be confirmed negative for the target pathogens by the 657 reference assay. 658

659 4. Sample size and sample panel composition for evaluation of performance characteristics:
660

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 22 of 41

Sample sizes of positive and negative samples of the RSV A/B against different values of 661 sensitivity and specificity are provided in Table 4. Sample sizes have been calculated assuming 662 95% level of significance, an absolute precision of 5%, and invalid test rate ≤5%. Appropriate 663 sample size has to be chosen from the tables according to the values of sensitivity and 664 specificity being claimed by the manufacturer. If a claimed sensitivity/specificity is not present 665 in the table, the manufacturer needs to consider the sample size associated with the largest 666 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as per 667 the next smaller value of the sensitivity/ specificity available in the table). For example, if a 668 manufacturer claims a sensitivity of 93%, they are required to use a sample size mentioned 669 against 90% sensitivity. Similarly, a claim of 87% specificity would require usage of the 670 sample size outlined for 85% specificity. Sample sizes for positive samples and their 671 composition for evaluating subtyping (RSV A/B) are provided in Table 5. Sample sizes are 672 calculated using the formulae: 673

674 𝑑2 x (1 −IR)
675 𝑑2 x (1 −IR)
676

677

678 · n (se) is the number of positive samples. 679 · n (sp) is the number of negative samples. 680 681 682 683 684 685 686

687

688 Table 4. Sample sizes per target pathogen (RSV A/B) for different values of sensitivity/ specificity 689 claimed by the manufacturer. 690 Sensitivity/ Specificity Sample size: Minimum number of positive samples¥ Composition of positive samples# Sample size: Minimum number of negative Minimum number of cross reactive* samples

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 23 of 41

samples (rounded)¥ among the negative samples 99% 16 (rounded to 20 for better Weak positive = 07 20 5 95% 77 (rounded to 80 for better Weak positive = 28 80 20 90% 146 (rounded to 155 for samples) Strong positive = 45 Moderate positive = 55 Weak positive =55 150 38 85% 207 (rounded to 215 for samples) Strong positive = 63 Moderate positive = 76 Weak positive = 76 210 53 80% 259 (rounded to 260 for samples) Strong positive = 78 Moderate positive = 91 Weak positive = 91 260 65 #Strong positive: (Ct value <25) Moderate positive: (Ct value between 25-30) Weak positive: (Ct value >30 and and ≤ 34)

¥ Nasopharyngeal/ oropharyngeal swabs in virus transport medium (VTM) to be used Samples positive for common respiratory viruses (such as Influenza (including its types and subtypes), SARS-CoV-2, Parainfluenza viruses, Adenoviruses, Rhinoviruses, common human coronaviruses), other than the ones targeted by the kit under evaluation. Equal distribution of cross-reactive viruses is desirable. It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 691 692 693 Table 5. Sample sizes for positive samples and their composition for evaluating subtyping
694 Sensitivity Sample size (per target pathogen) RSV A RSV B Minimum total positive samples Minimum number of nasopharyngeal swabs/ oropharyngeal swabs Minimum number of nasopharyngeal swabs/ oropharyngeal swabs 99% 20 20 20 40 95% 80 80 80 160 90% 150 150 150 300 85% 210 210 210 420 80% 260 260 260 520 *Combination of strong, moderate and weak positive samples should be considered as per the information provided in Table 4. It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 695 696 697

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 24 of 41

4.1 Repeatability testing will be performed on 3 positive (strong, moderate and weak 698 positive) and 3 negative samples (within the selected positive and negative samples) per 699 target pathogen 5 times (replicates of 5).
700 5. Methodology: 701 5.1 Samples should be tested in parallel with the Kit Under Evaluation and the reference 702 assay. The ICMR-NIV RT-qPCR assay for RSV or WHO Pre-Qualified/ US FDA/ ATAGI 703 Australia/ PMDA Japan approved kit will be considered as the reference assay for these 704 parameters. 705 5.2 The validation laboratory can use established total RNA / viral RNA extraction protocol 706 for the evaluation.
707 5.3 The instruction for the assay setup and the interpretation of the results will be as per 708 the protocol outlined by the manufacturer of the Kit Under Evaluation.
709 5.4 The results shall be compared with the reference assay for sensitivity and specificity 710 calculations. 711 5.5 If there is a discrepancy observed in the results with the index test, this discrepancy 712 should be taken as discordant. Repetition of the assay may introduce bias. If the reference 713 kit itself has failed, then these samples with discrepancies should be discarded, and new 714 well-characterized samples should be used instead.
715 True positive samples: These are samples positive by both reference assay and index test. 716 True negative samples: These are samples negative by both reference assay and index test. 717 718 index test. 719 720 index test. 721

722 5.6 The interpretation for internal control (preferably endogenous, or exogenous) will be 723 as per manufacturer's instruction.
724 5.7 PCR should be performed using IVD-approved machines. If any equipment(s) is 725 specified in the IFU of the index test, it should be used for the evaluation, and it should be 726 provided by the manufacturer if not available within the lab’s IVD evaluation scope.
727 Real-time closed systems/devices awaiting evaluation should be provided by the 728 manufacturer along with all necessary components, supplies and reagents. 729 The details on the Real-time Equipment used for validation should be recorded, including 730 calibration status. 731 The details on the Real-time Equipment used for validation should be recorded including 732 calibration status. 733

734

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 25 of 41

Cross-reactivity Analysis: 735 6.1 Objective:
736 To assess the primer-probe set for true detection of RSV and assess its cross-reactivity with 737 other respiratory viruses. 738 6.2 Methodology: 739 6.1.1 Potential cross-reactivity of the kit shall be ruled out by testing other respiratory 740 pathogen positive samples (N=30), with equal representation (n=5 each) of 741 samples positive for Influenza, SARS-CoV-2, Parainfluenza viruses, 742 Adenoviruses, Rhinoviruses, common human coronaviruses.* 743 6.1.2 Cross-reactivity will be assessed by comparing the results of these samples 744 using kit under evaluation and reference kit. 745 6.1.3 The kit targets should not show any amplification with other respiratory viruses 746 (ORVs). If amplification is observed for ORV then the kit will fail validation 747 and the same needs to be mentioned in the report.
748

For multiplex assays targeting influenza, SARS-CoV-2, and RSV detection, samples positive for 749 these viruses may be suitably interchanged for assessing cross-reactivity 750

751

752 7. Acceptance criteria for the kit:
753

754 Sensitivity for each pathogen/ type/ subtype: ≥95%
755 Specificity for each pathogen/ type/ subtype: ≥99%
756 757 758 To achieve at least the performance characteristics outlined in the acceptance criteria, ≥80 759 positive samples and ≥20 negative samples should be tested for evaluation for each 760 pathogen/ type/ subtype. 761

762 8. Repeatability Assessment:
763 8.1 Objectives: To assess the repeatability of the detection of SARS-CoV-2 using the kit 764 under evaluation
765

766 8.2 Sample size: Five replicate of 3 positive samples per target pathogen (strong, 767 moderate and weak positive) and five replicates of 3 negative samples per target pathogen 768 should be tested. 769

770 8.3 Result: Concordance should be 100% based on positive and negative test result (qualitative).
771

772 9. Precision (Reproducibility): 773

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 26 of 41

Lot to Lot Reproducibility 774

775 1.1 Objectives: To assess precision (reproducibility) among 3 different lots of the kit 776 under evaluation.
777 9.2 Sample size: Lot to lot variation testing: Three lots of an assay shall be evaluated. 778 Sample size for lot-to-lot reproducibility should be as follows:
779 780 and negative samples as calculated in the protocol.
781 • Second lot of the assay: should be tested on 25 samples (15 positive samples 782 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 783 samples).
784 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 785 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 786

787 9.3 Result: Concordance should be 100% based on positive and negative test result 788 (qualitative). 789

790 10. Internal Control Analysis: 791 10.1 Monitor the internal control (RNaseP or other endogenous housekeeping gene) to ensure 792 consistent extraction and amplification efficiency across samples and runs. 793 10.2 Ct-values of internal controls should be within the manufacturer’s prescribed limit. 794 10.3 Tests will be marked invalid if Ct-values are outside the prescribed limit.
795

796 11. Blinding of Laboratory Staff: 797 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should 798 be blinded to the status of the clinical samples. The PI of the evaluation exercise should 799 remain unblinded, i.e., privy to the status of the samples. Another senior laboratory staff 800 selected by the PI may remain unblinded and carry out coding of samples and dispensing 801 them into similar-looking vials to be used for testing, and maintaining the database of 802 results. Staff performing the reference test and the test under evaluation, interpretation of 803 the test result, and entering the results against the coded samples in the database, should 804 remain blinded to the status of samples till the completion of evaluation. The data should 805 be analyzed only by the PI of the evaluating lab. Refer to Fig. 1 in Protocol A. 806 12. Conclusion: 807 Based on the comprehensive evaluation conducted, the [Kit & Manufacturer’s Name] SARS-CoV- 808 2 RT-PCR Assay has been found [Satisfactory/Not Satisfactory] for its intended in vitro 809 diagnostic (IVD) use.
810

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 27 of 41

The assay demonstrates [Strengths/Concerns] in terms of sensitivity, specificity, and performance 811 characteristics compared to established reference IVD approved RT-PCR kits. 812

813 13. Publication Rights: 814 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead author(s). 815

816 817 818 acceptable.
819 820 821 822 823 824 825 826 827

828 14. Performance evaluation report format: 829 The performance evaluation report format (page 34) is designed for multiplex assays with several 830 targets. It should be modified and used accordingly for single plex assays/multiplex assays with 831 fewer targets. 832

833

834

835

836

837

838

839

840

841

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 28 of 41

Protocol D 842 Evaluation of performance characteristics of Molecular Kit detecting Influenza virus and 843 SARS-CoV-2 in multiplex format 844 To assess the performance of multiplex assays, Protocols A and B can be used as per kit format to 845 check the performance of each virus for its sensitivity and specificity assessment, including cross 846 reactivity, repeatability, reproducibility and Lot to lot variation.
847 A comprehensive report can be generated which will include sensitivity and specificity for all 848 targets.
849 Sample size for multiplex molecular assay (as per the scope outlined in the document) detecting 850 Influenza virus and SARS-CoV-2 in multiplex format is given below. All other 851 parameters/conditions outlined in the single plex protocols (Protocols A and B) are to be 852 essentially followed. 853

Sample size and sample panel composition for evaluation of performance 854 characteristics:
855 Sample sizes of positive and negative samples against different values of sensitivity and 856 specificity are provided in Table 6. Sample sizes have been calculated assuming 95% level of 857 significance, an absolute precision of 5%, and invalid test rate ≤5%. Appropriate sample size 858 has to be chosen from the tables according to the values of sensitivity and specificity being 859 claimed by the manufacturer. If a claimed sensitivity/specificity is not present in the table, the 860 manufacturer needs to consider the sample size associated with the largest 861 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as per 862 the next smaller value of the sensitivity/ specificity available in the table). For example, if a 863 manufacturer claims a sensitivity of 93%, they are required to use a sample size mentioned 864 against 90% sensitivity. Similarly, a claim of 87% specificity would require usage of the 865 sample size outlined for 85% specificity. Sample sizes are calculated using the formulae: 866

867 𝑑2 x (1 −IR)
868 𝑑2 x (1 −IR)
869

870

871 · n (se) is the number of positive samples. 872 · n (sp) is the number of negative samples. 873 874 875 876

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 29 of 41

877 878 879

880

881 Table 6. Sample sizes for different values of sensitivity/ specificity claimed by the 882 manufacturer. 883 Sensitivit y/ Specificit y Sample size for each of the 04 target pathogensa: Minimum number of positive samples¥ Composition of positive samples for each pathogen# Total number of positive samples (includin g all 04 pathogen s) Sample size: Minimum number of negative samples¥ Minimum number of cross reactive* samples among the negative samples 99% 16 (rounded to 20 for samples) Weak positive = 07 80 20 5 95% 77 (rounded to 80 for samples) Weak positive = 28 320 80 20 90% 146 (rounded to 155 for samples) Strong positive = 45 Moderate positive = 55 Weak positive =55 620 150 38 85% 207 (rounded to 215 for samples) Strong positive = 63 Moderate positive = 76 Weak positive = 76 860 210 53 80% 259 (rounded to 260 for samples) Strong positive = 78 Moderate positive = 91 Weak positive = 91 1040 260 65 aInfluenza A: (H1N1) pdm09, Influenza A/H3N2, Influenza B, and SARS CoV-2 #Strong positive: (Ct value <25) Moderate positive: (Ct value between 25-30) Weak positive: (Ct value >30 and and ≤ 34)

¥ Nasopharyngeal or oropharyngeal swabs in virus transport medium (VTM) to be used *Samples positive for common respiratory viruses (such as Parainfluenza viruses, Adenoviruses, Rhinoviruses, common human coronaviruses, RSV), other than the ones targeted by the kit under evaluation. Equal distribution of cross-reactive viruses is desirable.

For multiplex assays targeting influenza and SARS-CoV-2, samples positive for these viruses may be suitably interchanged for assessing cross-reactivity It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 884 885 886

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 30 of 41

Acceptance Criteria for the kit: 887 Sensitivity for each pathogen/ type/ subtype: ≥95%
888 Specificity for each pathogen/ type/ subtype: ≥99%
889 890 891 To achieve at least the performance characteristics outlined in the acceptance criteria, ≥80 892 positive samples and ≥20 negative samples should be tested for evaluation for each 893 pathogen/ type/ subtype. 894

895 896 897 acceptable.
898 899 900 901 902 903 904 905 906

907

908

909

910

911

912

913

914

915

916

917

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 31 of 41

Protocol E 918 Evaluation of performance characteristics of Molecular Kit detecting Influenza virus, 919 SARS-CoV-2 and Respiratory Syncytial Virus (RSV) in multiplex format 920 To assess the performance of multiplex assays, Protocols A, B or C can be used as per kit format 921 to check the performance of each virus for its sensitivity and specificity assessment, including 922 cross reactivity, repeatability, reproducibility and Lot to lot variation.
923 A comprehensive report can be generated which will include sensitivity and specificity for all 924 targets.
925 Sample size for multiplex molecular assay (as per the scope outlined in the document) detecting 926 Influenza virus, SARS-CoV-2 and Respiratory Syncytial Virus (RSV) in multiplex format is 927 given below. All other parameters/conditions outlined in the single plex protocols (Protocol A, B 928 and C) are to be essentially followed. 929

Sample size and sample panel composition for evaluation of performance 930 characteristics:
931 Sample sizes of positive and negative samples against different values of sensitivity and 932 specificity are provided in Table 7. Sample sizes have been calculated assuming 95% level of 933 significance, an absolute precision of 5%, and invalid test rate ≤5%. Appropriate sample size 934 has to be chosen from the tables according to the values of sensitivity and specificity being 935 claimed by the manufacturer. If a claimed sensitivity/specificity is not present in the table, the 936 manufacturer needs to consider the sample size associated with the largest 937 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as per 938 the next smaller value of the sensitivity/ specificity available in the table). For example, if a 939 manufacturer claims a sensitivity of 93%, they are required to use a sample size mentioned 940 against 90% sensitivity. Similarly, a claim of 87% specificity would require usage of the 941 sample size outlined for 85% specificity. Sample sizes are calculated using the formulae: 942

943 𝑑2 x (1 −IR)
944 𝑑2 x (1 −IR)
945

946

947 · n (se) is the number of positive samples. 948 · n (sp) is the number of negative samples. 949 950 951 952

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 32 of 41

953 954 955

956

957 Table 7. Sample sizes for different values of sensitivity/ specificity claimed by the 958 manufacturer. 959 Sensitivit y/ Specificit y Sample size for each of the 06 target pathogensa: Minimum number of positive samples¥ Composition of positive samples for each pathogen# Total number of positive samples (including all 06 pathogens) Sample size: Minimum number of negative samples¥ Minimu m number of cross reactive * sample s among the negativ e sample s 99% 16 (rounded to 20 for samples) Weak positive = 07 120 20 5 95% 77 (rounded to 80 for samples) Weak positive = 28 480 80 20 90% 146 (rounded to 155 for samples) Strong positive = 45 Moderate positive = 55 Weak positive =55 930 150 38 85% 207 (rounded to 215 for samples) Strong positive = 63 Moderate positive = 76 Weak positive = 76 1290 210 53 80% 259 (rounded to 260 for samples) Strong positive = 78 Moderate positive = 91 Weak positive = 91 1560 260 65 aInfluenza A: (H1N1) pdm09, Influenza A/H3N2, Influenza B, SARS CoV-2, RSV A, and RSV B #Strong positive: (Ct value <25) Moderate positive: (Ct value between 25-30) Weak positive: (Ct value >30 and and ≤ 34) ¥ Nasopharyngeal/ oropharyngeal swabs in virus transport medium (VTM) to be used

Samples positive for common respiratory viruses (such as Parainfluenza viruses, Adenoviruses, Rhinoviruses, common human coronaviruses), other than the ones targeted by the kit under evaluation. Equal distribution of cross-reactive viruses is desirable.

For multiplex assays targeting influenza, SARS-CoV-2, and RSV, samples positive for these viruses may be suitably interchanged for assessing cross-reactivity

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 33 of 41

It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 960 961 962

963 2. Acceptance Criteria for the kit: 964 Sensitivity for each pathogen/ type/ subtype: ≥95%
965 Specificity for each pathogen/ type/ subtype: ≥99%
966 967 968 To achieve at least the performance characteristics outlined in the acceptance criteria, ≥80 969 positive samples and ≥20 negative samples should be tested for evaluation for each 970 pathogen/ type/ subtype. 971

972 973 974 acceptable.
975 976 977 978 979 980 981 982 983

984

985

986

987

988

989

990

991

992

993

994

995

996

997

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 34 of 41

Performance evaluation report for Respiratory Virus in-vitro molecular diagnostic kit 998

999 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 Sample type Positive samples (provide details: strong, moderate, weak)
Negative samples (provide details, including cross reactivity panel)

1000

1001 i. Analytes/Pathogens targeted by the kit under evaluation: 1002 ………………………………………………… 1003 ii. ………………………………………………… 1004 iii. ………………………………………………… 1005 iv. ………………………………………………… 1006 v. ………………………………………………… 1007 vi. ………………………………………………… 1008 vii. ………………………………………………… 1009

1010

1011

1012

1013

1014

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 35 of 41

RESULTS INTERPRETATION 1015

1016

1017 SENSITIVITY AND SPECIFICITY FOR INDIVIDUAL VIRUS TARGETS 1018

Sensitivity and specificity for Influenza A (H1N1) pdm09
1019 Name of the Kit Under Evaluation

Reference assay Positive Negative Total Positive

Negative

Total

1020

Estimate (%) CI 95% Sensitivity

Specificity

1021 2. Sensitivity and specificity for Influenza A (H3N2)
1022 Name of the Kit Under Evaluation

Reference assay Positive Negative Total Positive

Negative

Total

1023

Estimate (%) CI 95% Sensitivity

Specificity

1024 3. Sensitivity and specificity for Influenza B (Victoria)
1025 Name of the Kit Under Evaluation

Reference assay Positive Negative Total Positive

Negative

Total

1026

Estimate (%) CI 95% Sensitivity

Specificity

1027

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 36 of 41

Sensitivity and specificity for Influenza B (Yamagata)
1028 Name of the Kit Under Evaluation

Reference assay Positive Negative Total Positive

Negative

Total

1029

Estimate (%) CI 95% Sensitivity

Specificity

1030 5. Sensitivity and specificity for SARS-CoV-2
1031 Name of the Kit Under Evaluation

Reference assay Positive Negative Total Positive

Negative

Total

1032

Estimate (%) CI 95% Sensitivity

Specificity

1033 6. Sensitivity and specificity for RSV A
1034 Name of the Kit Under Evaluation

Reference assay Positive Negative Total Positive

Negative

Total

1035

Estimate (%) CI 95% Sensitivity

Specificity

1036 7. Sensitivity and specificity for RSV B
1037 Name of the Kit Under Evaluation

Reference assay Positive Negative Total Positive

Negative

ICMR-CDSCO/IVD/GD/PROTOCOLS/05/2025 Page 37 of 41

Total

1038

Estimate (%) CI 95% Sensitivity

Specificity

1039 a. Cross-reactivity Analysis: 1040 b. Repeatability Assessment: 1041 c. Precision (Reproducibility): 1042 • Lot to Lot 1043

1044 Details of lots tested (3 lots to be tested): 1045

Lot No.:

Lot No:

Tested By: 1046 2. Lot No.:

Lot No:

Tested By: 1047 3. Lot No.:

Lot No:

Tested By: 1048

1049 ▪ Lot-to-lot variation was observed / not observed. 1050 d. Internal Control Analysis: 1051 Conclusion: Satisfactory / Not satisfactory
1052

1053 RECOMMENDATIONS: 1054 Suggestions for improvements or modifications (if applicable): 1055

1056 ▪ ICMR-CDSCO guidelines were followed for kit performance evaluation.
1057 This evaluation report is exclusively for____________________________________ In Vitro 1058 Molecular Diagnostic Kit manufactured by . 1059 Sensitivity and specificity have been assessed in controlled lab settings using the kits of the 1060 Lot number: 1061 i. Lot No., 1062 ii. Lot No., 1063 iii. Lot No._____, 1064

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Provided by the manufacturer, using ……. samples. Results should not be extrapolated to 1065 other sample types. 1066 DISCLAIMER: 1067

This validation process does not approve/disapprove the Kit design. 1068
This validation process does not certify user friendliness of the Kit. 1069
Influenza and SARS-CoV-2 are continuously evolving viruses and therefore primer 1070 probe sequences of the assay may require periodic updates, which will amount to a changed 1071 version of the assay. Re-validation is required for changed version of the assay, and needs 1072 to be considered while issuing license 1073

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1075 Signature of the Lab Manager

Signature of the Lab Director 1076

1077 Signature of Head of the Institute 1078

1079 Seal of Head of the Institute 1080

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1082 ****End of the Report 1083

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Annexure-1: Information on Operational and Test Performance Characteristics Required 1092 from Manufacturers 1093 The manufacturer should provide the following details about the IVD: 1094

Instructions for Use 1095
Scope of the IVD: to diagnose influenza and/or SARS-CoV-/RSV. 1096
Intended Use Statement 1097
Principle of the assay 1098
Intended testing population (cases of ARI/ILI/SARI) 1099
Intended user (laboratory professional and/or health care worker at point-of-care) 1100
Lot/batch No. 1101
Date of manufacture 1102
Date of Expiry 1103
Information on operational Characteristics 1104 i. Configuration of the kit/device 1105 ii. Requirement of any additional equipment, device 1106 iii. Requirement of any additional reagents 1107 iv. Operation conditions 1108 v. Storage and stability before and after opening 1109 vi. Internal control provided or not 1110 vii. Quality control and batch testing data 1111 viii. Biosafety aspects- waste disposal requirements 1112
Information on Test Performance Characteristics 1113 i. Type of sample-NP/OP swab, other respiratory specimen 1114 ii. Volume of sample 1115 iii. Any specific sample NOT to be tested 1116 iv. Any additional sample processing required 1117 v. Any additional device/consumable like sample transfer device, pipette, tube, etc required 1118

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vi. Name of analyte to be detected 1119 vii. Pathogens targeted by the kit 1120 viii. Time taken for testing 1121 ix. Time for result reading and interpretation 1122 x. Manual or automated(equipment)reading 1123 xi. Limit of detection 1124 xii. Diagnostic sensitivity 1125 xiii. Diagnostic specificity 1126 xiv. Stability and reproducibility 1127 xv. Training required for testing 1128 xvi. If yes, duration 1129 xvii. Details of Cut-off and /or Equivocal Zone for interpretation of test 1130 xviii. Interpretation of invalid and indeterminate results to be provided 1131 xix. It is recommended to provide data demonstrating the precision 1132 xx. Limit of detection 1133

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1 STANDARD PERFORMANCE 2 EVALUATION PROTOCOLS
3 DRAFT FOR STAKEHOLDER 4 COMMENTS 5

6 MALARIA IN-VITRO DIAGNOSTICS 7 ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025 8

10 AUGUST, 2025 New Delhi, India

Malaria IVD Performance Evaluation Protocols ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

11 Table of Contents 12 S.N. Topic Page Number 1.
Performance evaluation protocol for Malaria Rapid diagnostic test (RDT) kits 2 2.
Performance evaluation protocol for Malaria ELISA kits 14 3.
Performance evaluation protocol for Malaria real-time PCR kits 25 4.
Field evaluation protocol for combo Malaria Rapid Diagnostic Test (RDT) kits (detecting P vivax and P falciparum) 38 5.
Information on operational and test performance characteristics required from manufacturers 49

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

Performance evaluation protocol for Malaria Rapid diagnostic test (RDT) kits 33 I. Background:
34 CDSCO/ICMR, New Delhi have aimed to facilitate the evaluation and supply of Quality- 35 Assured in vitro Diagnostics (IVD) kits suitable for use in India. Hence, the following 36 guidelines shall establish the uniformity during the performance evaluation of IVD kits The 37 objective of performance evaluation is to independently validate the manufacturer’s claim 38 regarding in-vitro diagnostic kit (IVD) performance. 39 II. Purpose: 40 To evaluate the performance characteristics of rapid diagnostic test kit for the diagnosis of 41 malaria parasite using irreversibly de-identified leftover archived/ spiked clinical samples. 42 III. Requirements:
43 a) Instructions for use (IFU) 44 b) Supply of RDT kits under evaluation (with batch no.; lot no.; manufacturing and expiry 45 date and other required details).
46 c) Evaluation sites/laboratories (With required equipment) 47 d) Reference test kits 48 e) Characterised Evaluation panel 49 f) Laboratory supplies
50 IV. Ethical approvals:
51 Performance evaluation activities using irreversibly de-identified leftover clinical samples 52 are exempt from ethics approval as per ICMR’s Guidance on Ethical Requirements for 53 Laboratory Validation Testing, 2024.
54 Investigators are required to submit a self-declaration form, as outlined in the ICMR 55 guidelines, to the institutional authorities and ethics committee for information. 56 V. Procedure: 57

Study design/type: Diagnostic accuracy study using irreversibly de-identified leftover 58 clinical/spiked samples. 59
Preparation of Evaluation sites/laboratories:
60 Identified IVD kit evaluation laboratories should establish their proficiency 61 through:
62 a) Laboratory accreditation: Accreditation for at least one of the Quality management 63 systems (accreditation for Testing Lab / Calibration Lab (ISO: 17025), Medical 64 Lab (ISO: 15189), PT provider (ISO: 17043) or CDSCO approved Reference 65 laboratory.
66 b) It is recommended that malaria Medical Device Testing Labs (MDTLs) participate 67 in Quality Control exercises such as EQAP (External Quality Assurance 68 Programme). 69

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c) Staff training: All the staff involved in IVD kit evaluation should undergo hands 70 on training and competency testing on the following at referral level malaria labs 71 before initiation of MDTL activity:
72 ⮚ Preparation and characterization of evaluation panel for the respective IVD 73 kit. 74 ⮚ Management of RDT kits (specific for Plasmodium falciparum / Plasmodium 75 vivax) received for performance evaluation (Verification/Storage/Unpacking 76 etc.). 77 ⮚ Perform tests interpretation and documentation of results, and reporting. 78 ⮚ Data management and safety and confidentiality. 79 3. Preparation of QC panel members for Malaria RDT kit evaluation
80 To evaluate the performance of IVD kit, a well characterized species specific malaria 81 antigen sample panel is required. Statistically significant number of blood samples as 82 defined in this protocol should be collected from malaria confirmed cases in health 83 facilities, (as mentioned in Table 1). The panel should comprise positive and negative 84 samples as described in section 7.
85 The reference sample panel should be stored in appropriate storage conditions, and the 86 quality of the panel should be checked periodically through appropriate testing. 87 4. Reference assay:
88 WHO Pre-Qualified/ US FDA/ ATAGI Australia/ PMDA Japan approved RDT should 89 be used as reference standard. 90 All positive samples should be confirmed positive by the reference assay. 91 All negative samples should be confirmed negative by the reference assay. 92

93 5. Sample size and sample panel composition for performance evaluation:
94 Sample sizes of positive and negative samples of each species targeted by the kit against 95 different values of sensitivity and specificity are provided in Tables 1 and 2, with 96 recommended composition. Sample sizes have been calculated assuming 95% level of 97 significance, an absolute precision of 5%, and invalid test rate of 5%. Appropriate 98 sample size has to be chosen from the tables according to the values of sensitivity and 99 specificity being claimed by the manufacturer. If a claimed sensitivity/specificity is not 100 present in the table, the manufacturer needs to consider the sample size associated with 101 the largest sensitivity/specificity provided in the table that is smaller to the claimed 102 value (that is, as per the next smaller value of the sensitivity/ specificity available in the 103 table). For example, if a manufacturer claims a sensitivity of 93%, they are required to 104 use a sample size mentioned against 90% sensitivity. Similarly, a claim of 87% 105 specificity would require usage of the sample size outlined for 85% specificity.
106

107

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Sample sizes are calculated using the formulae: 108

109 𝑑2 x (1 −IR)
110 𝑑2 x (1 −IR)
111

112

113 · n (se) is the minimum number of positive samples. 114 · n (sp) is the minimum number of negative samples. 115 · Z2 is the critical value from the standard normal distribution 116 corresponding to the desired confidence level (95% CI corresponds to Z2 117 =1.96). 118 119 120 121 122

123

124 Table 1. Positive sample sizes (per species) and composition for different values of 125 sensitivity claimed by the manufacturer for evaluation of Pf (single/combo RDT) or Pv 126 (single/combo RDT) 127 Sensitivity Sample size: Minimum number of positive samples # Composition of positive samples 99% 16 (rounded to 20 for better distribution of samples) Weak positive = 07 95% 77 (rounded to 80 for better distribution of samples) Weak positive = 28 90% 146 (rounded to 155 for better Strong positive = 45 Moderate positive = 55 Weak positive =55 85% 207 (rounded to 215 for better Strong positive = 63 Moderate positive = 76 Weak positive = 76 80% 259 (rounded to 260 for better Strong positive = 78 Moderate positive = 91 Weak positive = 91 75% 305 (rounded to 310 for better Strong positive = 92 Moderate positive = 109 Weak positive = 109

128

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#It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and 129 specificity; however, a higher sample size is suggested to ensure adequate power of the study 130 in case the kit falls short of claimed performance characteristics. 131

132 Table 2. Negative sample sizes and composition for different values of specificity 133 claimed by the manufacturer for evaluation of Pf (single/combo RDT) or Pv 134 (single/combo RDT) 135 Specificity Sample size: Minimum number of negative samples # Composition of negative samples# 99% 16 (rounded to 20) Dengue NS1/IgM positive samples: 03 Chikungunya IgM positive samples:03 Serum reactive for RA factor – low positive and high positive:02 Serum reactive for TPHA/other specific test for syphilis:02 Healthy controls from endemic regions: 10

95% 77 (rounded to 80) Dengue NS1/IgM positive samples: 10 Chikungunya IgM positive samples:10 Serum reactive for RA factor – low positive and high positive:10 Serum reactive for TPHA/other specific test for syphilis:10 Healthy controls from endemic regions: 40

90% 146 (rounded to 150) Dengue NS1/IgM positive samples: 18 Chikungunya IgM positive samples:18 Serum reactive for RA factor – low positive and high positive:18 Serum reactive for TPHA/other specific test for syphilis:18 Healthy controls from endemic regions: 78

85% 207 (rounded to 210) Dengue NS1/IgM positive samples: 26 Chikungunya IgM positive samples:26 Serum reactive for RA factor – low positive and high positive:26 Serum reactive for TPHA/other specific test for syphilis:26 Healthy controls from endemic regions: 106

80% 259 (rounded to 260) Dengue NS1/IgM positive samples: 35 Chikungunya IgM positive samples:35 Serum reactive for RA factor – low positive and high positive:30 Serum reactive for TPHA/other specific test for syphilis:30 Healthy controls from endemic regions: 130

136 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and 137 specificity; however, a higher sample size is suggested to ensure adequate power of the study 138 in case the kit falls short of claimed performance characteristics. 139 Sample panel composition: 140

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Positive samples: Malaria positive samples should be obtained from health facilities 141 (tertiary care centers and their linked hospitals, private clinics, field practice areas 142 etc.) and confirmed using PCR (Snounou protocol/FDA approved assay). 143 Malaria samples confirmed positive by PCR should be characterized for parasite load 144 on in-house calibrated equipment using blood smear microscopy and ELISA. Samples 145 with analyte values satisfying the range of acceptance criteria (as mentioned in this 146 document) should be included in the positive sample panel for the evaluation of malaria 147 RDT kits.
148 For the RDT kits which have other antigen/antibody as target analyte (for which limits 149 of detection have not been established), characterization of samples should be 150 performed on calibrated equipment, leading to their classification as low and high 151 parasitemic samples, which should then be used for performance evaluation of the 152 assay.
153

154 Range of Parasitemia: Panel members should have low (≤200 parasites per microliter) 155 to high (≥2000 parasites per microliter) range of Plasmodium falciparum, P. vivax 156 and/or other Plasmodium species, as obtained from ELISA results. Characterized 157 panels must contain equal number of samples of both low and high parasitemia. 158 Consistent ELISA quantification results should be obtained in ≥3 runs of ELISA 159 experiments performed for each of the three antigens (PfHRP2, LDH and aldolase) with 160 the results obtained at the 200 p/µL and the 2,000 p/µL being consistent with each other 161 as well (factor of roughly 10 between results). The limit of detection of Pfhrp2 is 5-10 162 ng/ µL, and PvLDH is 15-45 ng/ µL.
163 ** It should be noted that no such limit of detection is defined for aldolase. Where values/standard 164 reference assay not available, standard procedure on calibrated equipment will be followed for obtaining 165 results. 166

167 6. Test reproducibility: 168

169 A. Sample size for lot-to-lot reproducibility 170

171 Three lots of an assay shall be evaluated. The first lot shall be evaluated on the entire 172 panel of samples (statistically significant sample size). For the subsequent two lots, 25 173 samples should be used for evaluation (15 positive samples including 10 weak positive 174 samples and 5 moderate/strong positive samples, and 10 negative samples).
175 Refer the flowchart below (Fig. 1): 176

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178

179

180 B. Reader-to-reader reproducibility: 25 samples (15 positive samples including 10 181 weak positive samples and 5 strong/moderate positive samples, and 10 negative 182 samples) need to be tested by at least 2 trained personnel. Agreement should be 183 100%. 184

185 Note: Testing Methodology 186 Read the instructions for use (IFU) thoroughly. Take out the required number of RDTs kits 187 from the recommended storage conditions. Bring RDTs to room temperature (20°C - 30°C) 188 and thaw the required number of QC/sample panel aliquots for a minimum of 20 minutes to 189 maximum 60 minutes before performing the test. Note that more than one aliquot may be 190 needed for the testing of each sample. Record the results of the performance evaluation on the 191 recommended report format ( Annexure 1). 192 7. Evaluation method: 193 The reference assay and the index test should be run on the sample panel in parallel. 194

195 8. Interpretation of results:
196 Results should be interpreted as per the IFU of the reference assay and the index test.
197

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198 9. Resolution of discrepant results: 199 True positive samples: These are samples positive by both reference assay and index 200 test. 201 True negative samples: These are samples negative by both reference assay and index 202 test. 203 204 index test. 205 206 index test. 207

208 10. Acceptance criteria1: 209 Expected sensitivity: ≥75% for P. vivax and ≥95% for P. falciparum 210 Expected specificity: ≥90% for P. vivax and ≥95% for P. falciparum 211 212 213

214 To achieve at least the performance characteristics outlined in the acceptance criteria, 215 ≥310 positive samples and ≥150 negative samples should be tested for P vivax, and ≥80 216 positive samples and ≥80 negative samples should be tested for P falciparum. 217

218 11. Blinding of laboratory staff 219 To ensure rigor of the evaluation process, laboratory staff performing the evaluation 220 should be blinded to the status of the clinical samples. The PI of the evaluation exercise 221 should remain unblinded, i.e., privy to the status of the samples. Another senior 222 laboratory staff selected by the PI may remain unblinded and carry out coding of 223 samples and dispensing them into similar-looking vials to be used for testing, and 224 maintaining the database of results. Staff performing the reference test and the test 225 under evaluation, interpretation of the test result, and entering the results against the 226 coded samples in the database, should remain blinded to the status of samples till the 227 completion of evaluation. The data should be analyzed only by the PI of the evaluating 228 lab. Refer to Fig. 2. 229

230 Fig.2: Blinding in evaluation exercise 231

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232

233 12. Publication Rights 234 The PI(s) of the evaluating labs shall retain publication rights to the evaluation as lead 235 author(s). 236 After following due procedure as defined in this document, once any kit is found to be Not of 237 Standard Quality, thereafter, no request for repeat testing of the same kit will be acceptable. 238 239 entertained if valid proof of change in the kit composition is submitted. 240

241 After following due procedure as defined in this document, once any kit is found to be 242 Not of Standard Quality, thereafter, no request for repeat testing of the same kit will be 243 acceptable.
244 Any request of re-validation from the same manufacturer for the same test type will only 245 be entertained after a minimum of 3 months and only if a high-level technical summary 246 of modifications or functional improvements to the kit design is submitted, without 247 explicit disclosure of proprietary information. 248 Clinical samples are precious, therefore, repeat evaluation of a kit using the same/ 249 different well-characterized sample panel at a different laboratory may be considered 250 only for kits which claim high performance characteristics (sensitivity and specificity 251 95% and above), but which fail the performance evaluation by a margin of 5%. 252

253 VI. References: 254

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Ministry of Health and Family Welfare. Guidelines for Bivalent RDT. Available at: 255 guidelines-for-bivalent-rdt.pdf (mohfw.gov.in) 256
World Health Organization. Malaria Rapid Diagnostic Test Performance - Results of 257 WHO product testing of malaria RDTs: round 8 (2016–2018): Available at: 258 https://iris.who.int/bitstream/handle/10665/276190/9789241514965- 259 eng.pdf?sequence=1
260
Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Do Rosario VE, et al. High 261 sensitivity of detection of human malaria parasites by the use of nested polymerase 262 chain reaction. Molecular and Biochemical Parasitology. 1993;61:315–20. 263
Krishna S, Bharti PK, Chandel HS, Ahmad A, Kumar R, Singh PP, et al. Detection of 264 Mixed Infections with Plasmodium spp. by PCR, India, 2014. Emerg Infect Dis. 265 2015;21(10):1853-7. 266

267

268 VII. Performance evaluation report format

269

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REPORT FORMAT 270 Name of the Laboratory 271 Name of the Institute, (with station) 272 Certificate of Analysis 273 File No.: _________________________________________ 274 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Sample type Positive samples (provide details: strong, moderate, weak)

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Negative samples (provide details, including cross reactivity panel)

275

276 Results: 277

278

Reference assay ……….……………… (name)

Positive Negative Total Name of index malaria RDT
Positive

Negative

Total

279

280

Estimate (%) 95% CI Sensitivity

Specificity

281 ● Details of cross reactivity with other agents:
282 ● Conclusions: 283 o Sensitivity, specificity 284 o Performance: Satisfactory / Not Satisfactory 285 (Sensitivity and specificity have been assessed in controlled lab setting on serum samples only, 286 using kits provided by the manufacturer from the batch mentioned above. Results should not 287 be extrapolated for any other sample type.) 288 Disclaimers 289

This validation process does not approve / disapprove the kit design 290
This validation process does not certify user friendliness of the kit / assay 291

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Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by …………… 292 (Supplied by ……….) 293

294 Evaluation Done on …………………… 295 Evaluation Done by …………………………. 296

297 Signature of Director/ Director-In-charge ……………………

Seal 298 ……………………………………… 299 ****End of the Report 300

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Performance evaluation protocol for Malaria ELISA kits 323 I. Background:
324 CDSCO/ICMR, New Delhi, have aimed to facilitate the evaluation and supply of Quality- 325 Assured In Vitro Diagnostics kits suitable for use in India. Hence, the following guidelines 326 shall establish the uniformity during the performance evaluation of IVD kits . The objective 327 of performance evaluation is to independently validate the manufacturer’s claim regarding 328 in-vitro diagnostic kit (IVD) performance. 329 II. Purpose:
330 To evaluate the performance characteristics of malaria ELISA kits for the diagnosis of 331 malaria parasite infection using irreversibly de-identified leftover archived/ spiked clinical 332 samples. The malaria ELISA kits are designed to detect antigens (hrp2, LDH, aldolases) 333 occurring in subjects infected with species specific (P. falciparum, P. vivax) and stage 334 specific antibodies (MSP1, MSP3, CSP, EBA175 etc.- parasite markers for the purpose of 335 sero-survey). 336 III. Requirements:
337 a) Instructions for use (IFU) 338 b) Supply of ELISA kits under evaluation (with batch no./lot no. expiry date & required 339 details). In case the kit to be evaluated is designed to work in a closed system format, 340 the manufacturer needs to supply the required equipment. 341 c) Evaluation sites/laboratories (With required equipment) 342 d) Reference test kits 343 e) Characterised Evaluation panel 344 f) Laboratory supplies
345 IV. Ethical approvals:
346 Performance evaluation activities using irreversibly de-identified leftover clinical 347 samples are exempt from ethics approval as per ICMR’s Guidance on Ethical 348 Requirements for Laboratory Validation Testing, 2024.
349 Investigators are required to submit a self-declaration form, as outlined in the ICMR 350 guidelines, to the institutional authorities and ethics committee for information. 351 V. Procedure: 352

Study design/type: Diagnostic accuracy study using irreversibly de-identified leftover 353 clinical samples. 354 355 Identified ELISA kit evaluation laboratories should establish their proficiency 356 through
357 a) Laboratory accreditation: Accreditation for at least one of the Quality management 358 systems (accreditation for Testing Lab / Calibration Lab (ISO: 17025), Medical 359

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Lab (ISO:15189), PT provider ISO: 17043 or CDSCO approved Reference 360 laboratory. 361 b) It is recommended that malaria Medical Device Testing Labs (MDTLs) participate 362 in Quality Control exercises such as EQAP (External Quality Assurance 363 Programme). 364 c) Staff training: All the staff involved in ELISA kit evaluation should undergo 365 hands on training and competency testing on the following at referral level malaria 366 labs before initiation of MDTL activity:
367 ➢ Preparation and characterization of evaluation panel for the respective 368 ELISA kit. 369 ➢ Management of malaria ELISA kits received for performance evaluation 370 (Verification/Storage/Unpacking etc). 371 ➢ Perform tests , interpretation and documentation of results and reporting. 372 ➢ Data management and safety and confidentiality 373 3. Reference sample panel:
374 To evaluate the performance of ELISA kit a well characterised malaria stage specific 375 antigens/species specific antibody ELISA evaluation sample panel is required. In the 376 absence of WHO Pre-Qualified/US FDA/ ATAGI Australia/ PMDA Japan approved 377 malaria ELISA assay, it is recommended that performance evaluation of ELISA assays 378 be performed on a rigorously well characterized panel of positive and negative samples.
379 A statistically significant number of sera samples should be collected from malaria 380 confirmed cases from health facilities. All samples should be further confirmed by PCR 381 assay (Snounou protocol/FDA approved assay). 382 A. Malaria samples confirmed positive by PCR should be characterized for parasite load 383 on in-house calibrated equipment using ELISA. Samples with analyte values satisfying 384 the range of acceptance criteria (as mentioned in this document) should be included in 385 the positive sample panel for the evaluation of malaria RDT kits.
386 For those kits which have other antigen/antibody as target analyte (for which limits of 387 detection have not been established), characterization of samples for that analyte 388 should be performed on calibrated equipment, leading to their classification as low and 389 high parasitemic samples, which will then be used for performance evaluation of the 390 assay.
391

392 Range of Parasitemia: Panel members should have low (≤200 parasites per microliter) 393 to high (≥2000 parasites per microliter) range of Plasmodium falciparum, P. vivax 394 and/or other Plasmodium species, as obtained from ELISA results. Characterized 395 panels must contain equal number of samples of both low and high parasitemia. 396 Consistent ELISA quantification results should be obtained in ≥3 runs of ELISA 397 experiments performed for each of the three antigens (PfHRP2, LDH and aldolase – 398 recombinantly expessed proteins) with the results obtained at the 200 p/µL and the 399

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2,000 p/µL being consistent with each other as well (factor of roughly 10 between 400 results). The limit of detection of Pfhrp2 is 5-10 ng/ µL, and Pvldh is 15-45 ng/ µL.
401 ** It should be noted that no such limit of detection is defined for aldolase. Where values/standard 402 reference assay not available, standard procedure on calibrated equipment will be followed for obtaining 403 results. 404 The above-mentioned activities should not be performed with spiked/contrived samples. 405 Equal representation of samples positive for Plasmodium (P.falciparum /P.vivax) species 406 preferred. 407 B. Negative panel should constitute malaria negative samples (confirmed by PCR) as 408 described in point 6B. 409 The reference sample panel should be stored in appropriate storage conditions, and the quality 410 of the panel should be checked periodically with appropriate tests (including parasite culture) 411 as needed. 412 Malaria positive samples should be obtained from health facilities, including tertiary care 413 centers and their linked hospitals, private clinics, field practice areas etc.
414 Wherever any WHO Pre-Qualified/ US FDA/ ATAGI Australia/ PMDA Japan approved 415 assay is available, it should be used as reference standard. 416

417 Sample size and sample panel composition for performance evaluation: Sample sizes 418 of positive and negative samples of each species targeted by the kit against different values 419 of sensitivity and specificity are provided in Table 1 and Table 2, with recommended 420 composition. Sample sizes have been calculated assuming 95% level of significance and 421 an absolute precision of 5%. Appropriate sample size has to be chosen from the tables 422 according to the values of sensitivity and specificity being claimed by the manufacturer. If 423 a claimed sensitivity/specificity is not present in the table, the manufacturer needs to 424 consider the sample size associated with the largest sensitivity/specificity provided in the 425 table that is smaller to the claimed value (that is, as per the next smaller value of the 426 sensitivity/ specificity available in the table). For example, if a manufacturer claims a 427 sensitivity of 93%, they are required to use a sample size mentioned against 90% 428 sensitivity. Similarly, a claim of 87% specificity would require usage of the sample size 429 outlined for 85% specificity. Sample sizes are calculated using the formulae: 430

431 𝑑2

432 𝑑2

433

434

435 436 437

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· Z2 is the critical value from the standard normal distribution 438 corresponding to the desired confidence level (95% CI corresponds to Z2 439 =1.96). 440 441 442 443

444 Table 1. Positive sample sizes (per species) and composition for different values of sensitivity 445 claimed by the manufacturer for evaluation of Pf (single/combo) or Pv (single/combo) ELISA 446 Sensitivity Sample size: Minimum number of positive samples# Composition of positive samples 99% 16 (rounded to 20 for better distribution of samples) Weak positive = 07 95% 73 (rounded to 80 for better distribution of samples) Weak positive = 28 90% 139 (rounded to 140 for better Strong positive = 42 Moderate positive = 49 Weak positive = 49 85% 196 (rounded to 200 for better Strong positive = 60 Moderate positive = 70 Weak positive = 70 80% 246 (rounded to 255 for better Strong positive = 75 Moderate positive = 90 Weak positive = 90 75% 289 (rounded to 295 for better Strong positive = 87 Moderate positive = 104 Weak positive = 104 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and 447 specificity; however, a higher sample size is suggested to ensure adequate power of the study 448 in case the kit falls short of claimed performance characteristics. 449 Table 2. Negative sample sizes and composition for different values of specificity claimed by 450 the manufacturer for evaluation of Pf (single/combo) or Pv (single/combo) ELISA 451 Specificity Sample size: Minimum number of negative samples # Composition of negative samples 99% 16 (rounded to 20) Dengue NS1/IgM positive samples: 03 Chikungunya IgM positive samples:03 Serum reactive for RA factor – low positive and high positive:02 Serum reactive for TPHA/other specific test for syphilis:02 Healthy controls from endemic regions: 10

95% 73 (rounded to 80) Dengue NS1/IgM positive samples: 10 Chikungunya IgM positive samples:10

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Serum reactive for RA factor – low positive and high positive:10 Serum reactive for TPHA/other specific test for syphilis:10 Healthy controls from endemic regions: 40

90% 139 (rounded to 140) Dengue NS1/IgM positive samples: 18 Chikungunya IgM positive samples:18 Serum reactive for RA factor – low positive and high positive:18 Serum reactive for TPHA/other specific test for syphilis:18 Healthy controls from endemic regions: 68

85% 196 (rounded to 200) Dengue NS1/IgM positive samples: 25 Chikungunya IgM positive samples:25 Serum reactive for RA factor – low positive and high positive:25 Serum reactive for TPHA/other specific test for syphilis:25 Healthy controls from endemic regions: 100

80% 246 (rounded to 250) Dengue NS1/IgM positive samples: 30 Chikungunya IgM positive samples:30 Serum reactive for RA factor – low positive and high positive:30 Serum reactive for TPHA/other specific test for syphilis:30 Healthy controls from endemic regions: 130

#It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and 452 specificity; however, a higher sample size is suggested to ensure adequate power of the study 453 in case the kit falls short of claimed performance characteristics. 454

455

456 4. Sample size for lot-to-lot reproducibility 457 Three lots of an assay shall be evaluated. The first lot shall be evaluated on the entire 458 panel of samples (statistically significant sample size). For the subsequent two lots, 25 459 samples should be used for evaluation (15 positive samples including 10 weak positive 460 samples and 5 moderate/strong positive samples, and 10 negative samples).
461 462

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463

464

465 5. Evaluation Methodology:
466 The index test should be tested on a rigorously well-characterized panel of samples 467 from confirmed malaria positive and negative cases, which are further tested for the 468 presence of malaria parasite using the Snounou protocol.
469

470 6. Interpretation of results:
471 Results should be interpreted as per the IFU of the reference assay.
472

473 7. Resolution of discrepant results: 474 True positive samples: These are well-characterized samples from confirmed malaria 475 positive cases, which are also positive by the index test. 476 True negative samples: These are well-characterized samples from confirmed malaria 477 negative cases, which are also negative by the index test. 478 False positive samples: These are well-characterized samples from confirmed malaria 479 negative cases, which are positive by the index test. 480 False negative samples: These are well-characterized samples from confirmed malaria 481 positive cases, which are negative by the index test. 482

483

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484 8. Acceptance Criteria: 485

486 Type of assay Acceptance criteria Minimum no. of samples needed to achieve at least the performance characteristics outlined in the acceptance criteria

Malaria antibody ELISA Sensitivity: ≥90%
Specificity: ≥95% Minimum no. of Positive samples = 140

Minimum no. of Negative samples = 80 Pv ELISA Sensitivity: ≥75%
Specificity: ≥95% Minimum no. of Positive samples = 295

Minimum no. of Negative samples = 80 Pf ELISA Sensitivity: ≥90%
Specificity: ≥95% Minimum no. of Positive samples = 140

Minimum no. of Negative samples = 80 487

488

489 9. Blinding of laboratory staff 490 To ensure rigor of the evaluation process, laboratory staff performing the evaluation 491 should be blinded to the status of the clinical samples. The PI of the evaluation exercise 492 should remain unblinded, i.e., privy to the status of the samples. Another senior 493 laboratory staff selected by the PI may remain unblinded and carry out coding of samples 494 and dispensing them into similar-looking vials to be used for testing, and maintaining 495 the database of results. Staff performing the reference test and the test under evaluation, 496 interpretation of the test result, and entering the results against the coded samples in the 497 database, should remain blinded to the status of samples till the completion of 498 evaluation. The data should be analyzed only by the PI of the evaluating lab. Refer to 499 Fig. 2. 500

501 502

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503

504 10. Publication Rights 505 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead 506 author(s). 507 After following due procedure as defined in this document, once any kit is found to be 508 Not of Standard Quality, thereafter, no request for repeat testing of the same kit will be 509 acceptable.
510 Any request of re-validation from the same manufacturer for the same test type will only 511 be entertained after a minimum of 3 months and only if a high-level technical summary 512 of modifications or functional improvements to the kit design is submitted, without 513 explicit disclosure of proprietary information. 514 Clinical samples are precious, therefore, repeat evaluation of a kit using the same/ 515 different well-characterized sample panel at a different laboratory may be considered 516 only for kits which claim high performance characteristics (sensitivity and specificity 517 95% and above), but which fail the performance evaluation by a margin of 5%. 518

519

520 VI. References: 521

Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Do Rosario VE, et al. High 522 sensitivity of detection of human malaria parasites by the use of nested polymerase 523 chain reaction. Molecular and Biochemical Parasitology. 1993;61:315–20. 524

525 VII. Performance evaluation report format 526

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REPORT FORMAT 527 Name of the Laboratory 528 Name of the Institute, (with station) 529 Certificate of Analysis 530 File No.: _________________________________________ 531 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Sample type Positive samples (provide details: strong, moderate, weak)

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Negative samples (provide details, including cross reactivity panel)

532

533 Results: 534

535

Samples with confirmed disease status (Further confirmed by Snounou protocol/ FDA approved assay)

Positive Negative Total Name of malaria ELISA kit Positive

Negative

Total

536

537

Estimate (%) 95% CI Sensitivity

Specificity

538 ● Details of cross reactivity with other agents:
539 ● Conclusions: 540 o Sensitivity, specificity 541 o Performance: Satisfactory / Not Satisfactory 542 (Sensitivity and specificity have been assessed in controlled lab setting on ……. samples only, 543 using kits provided by the manufacturer from the batch mentioned above. Results should not 544 be extrapolated for any other sample type.) 545 Disclaimers 546 547 548

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Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by …………… 549 (Supplied by ……….) 550

551 552 553

554 Signature of Director/ Director-In-charge ……………………

Seal 555 ……………………………………… 556 ****End of the Report 557

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Performance evaluation protocol for Malaria real-time PCR kits 580 I. Background:
581 CDSCO/ICMR, New Delhi, have aimed to facilitate the evaluation and supply of Quality- 582 Assured Diagnostics kits appropriate for use in India. Hence the following guidelines shall 583 establish the uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The 584 performance evaluation is to independently verify the manufacturer’s claim regarding IVD kit 585 performance. 586 II. Purpose:
587 To evaluate the performance characteristics of Malaria real-time PCR (RT-PCR) kits using 588 irreversibly de-identified leftover archived/ spiked clinical samples. 589 III. Requirements:
590 1. Instructions for use (IFU) 591 2. Supply of kits under evaluation (with batch no. and lot no. ; Manufacturing and 592 Expiry and other required details). If the kit to be evaluated works in a closed 593 system format, the manufacturer needs to supply the required equipment. 594 3. Evaluation sites/laboratories (With required equipment) 595 4. Reference test kits 596 5. Characterised Evaluation panel 597 6. Laboratory supplies
598

599 IV. Ethical approvals: 600 Performance evaluation activities using irreversibly de-identified leftover clinical samples 601 are exempt from ethics approval as per ICMR’s Guidance on Ethical Requirements for 602 Laboratory Validation Testing, 2024.
603 Investigators are required to submit a self-declaration form, as outlined in the ICMR 604 guidelines, to the institutional authorities and ethics committee for information. 605

606 V. Procedure: 607

Study design/type: Diagnostic accuracy study using irreversibly de-identified leftover 608 clinical/spiked samples. 609 610 Identified IVD kit evaluation laboratories should establish their proficiency 611 through
612 a) Laboratory accreditation: Accreditation for at least one of the Quality management systems 613 (accreditation for Testing Lab / Calibration Lab (ISO: 17025), Medical Lab (ISO: 15189), 614 PT provider (ISO: 17043) or CDSCO approved Reference laboratory. 615 b) It is recommended that malaria Medical Device Testing Labs (MDTLs) participate in 616 Quality Control exercises such as EQAP (External Quality Assurance Programme). 617

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c) Staff training: All the staff involved in IVD kit evaluation should undergo hands-on 618 training and competency testing on the following at referral level malaria labs before 619 initiation of MDTL activity:
620 ➢ Preparation and characterization of evaluation panel for the respective IVD kit. 621 ➢ Management of RDT kits (specific for Plasmodium falciparum / Plasmodium vivax) 622 received for performance evaluation (Verification/Storage/Unpacking etc.). 623 ➢ Perform tests interpretation and documentation of results, and reporting. 624 ➢ Data management and safety and confidentiality. 625

Preparation of evaluation sample panel for Malaria 626 To evaluate the performance of malaria RT-PCR IVD kit, a well characterized species specific 627 malaria whole genome panel is required. Hence, statistically significant number of whole blood 628 samples should be collected from malaria confirmed cases. The panel should comprise positive 629 and negative samples as described in section 8. 630 The reference sample panel should be stored in appropriate storage conditions, and the quality 631 of the panel should be checked periodically with appropriate tests (including parasite culture) 632 as needed. 633 Malaria positive samples should be obtained from health facilities, including tertiary care 634 centers and their linked hospitals, private clinics, field practice areas etc.
635

636 2. DNA extraction 637 DNA extraction should be performed using a standard protocol/kit as recommended by the 638 manufacturer, or fully automated DNA extractor may be used (as per manufacturer’s 639 instruction and compatible reagent kits).
640 Note: If the manufacturer of the index test recommends a specific DNA extraction kit, it needs 641 to be provided by the manufacturer, if the evaluation lab is unable to procure the same. 642 3. Real-time PCR system: 643 PCR should be performed using IVD-approved machines. If any equipment(s) is specified in 644 the IFU of the index test, it should be used for the evaluation, and it should be provided by the 645 manufacturer if not available within the lab’s IVD evaluation scope.
646 Real-time closed systems/devices awaiting evaluation should be provided by the manufacturer 647 along with all necessary components, supplies and reagents. 648 4. Internal Control/Extraction Control: 649 The index test must have an internal control (housekeeping gene), with or without an extraction 650 control. 651 5. Reference assay:
652

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Two WHO Pre-Qualified/ US FDA/ ATAGI Australia/ PMDA Japan-approved malaria RT- 653 PCR assays (or one FDA-approved assay and the Snounou protocol) should be used as 654 reference assays for the characterization of samples, with 100% agreement between their 655 results.
656 All positive samples should be confirmed positive by the reference assay(s). 657 All negative samples should be confirmed negative by the reference assay(s). 658

659 6. Sample size and sample panel composition for performance evaluation:
660 Sample sizes of positive and negative samples of each species targeted by the kit against 661 different values of sensitivity and specificity are provided in Table 1 and Table 2, with 662 recommended composition. Sample sizes have been calculated assuming 95% level of 663 significance, an absolute precision of 5%, and invalid test rate of 5%. Appropriate sample 664 size has to be chosen from the tables according to the values of sensitivity and specificity 665 being claimed by the manufacturer. If a claimed sensitivity/specificity is not present in the 666 table, the manufacturer needs to consider the sample size associated with the largest 667 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as 668 per the next smaller value of the sensitivity/ specificity available in the table). For example, 669 if a manufacturer claims a sensitivity of 93%, they are required to use a sample size 670 mentioned against 90% sensitivity. Similarly, a claim of 87% specificity would require 671 usage of the sample size outlined for 85% specificity. Sample sizes are calculated using the 672 formulae: 673

674 𝑑2 x (1 −IR)
675 𝑑2 x (1 −IR)
676

677

678 679 680 · Z2 is the critical value from the standard normal distribution 681 corresponding to the desired confidence level (95% CI corresponds to Z2 682 =1.96). 683 684 685 686 687

688

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689

690 Table 1. Positive sample sizes (per species) and composition for different values of sensitivity 691 claimed by the manufacturer for evaluation of Pf (single/combo RDT) or Pv (single/combo 692 RDT) 693 Sensitivity Sample size: Minimum number of positive samples# Composition of positive samples 99% 16 (rounded to 20 for better distribution of samples) Weak positive = 07 95% 77 (rounded to 80 for better distribution of samples) Weak positive = 28 90% 146 (rounded to 155 for better Strong positive = 45 Moderate positive = 55 Weak positive =55 85% 207 (rounded to 215 for better Strong positive = 63 Moderate positive = 76 Weak positive = 76 80% 259 (rounded to 260 for better Strong positive = 78 Moderate positive = 91 Weak positive = 91 75% 304 (rounded to 310 for better Strong positive = 92 Moderate positive = 109 Weak positive = 109 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and 694 specificity; however, a higher sample size is suggested to ensure adequate power of the study 695 in case the kit falls short of claimed performance characteristics. 696 Table 2. Negative sample sizes and composition for different values of specificity claimed by 697 the manufacturer for evaluation of Pf (single/combo RDT) or Pv (single/combo RDT) 698 Specificity Sample size: Minimum number of negative samples# Composition of negative samples 99% 16 (rounded to 20) Dengue NS1/IgM positive samples: 03 Chikungunya IgM positive samples:03 Serum reactive for RA factor – low positive and high positive:02 Serum reactive for TPHA/other specific test for syphilis:02 Healthy controls from endemic regions: 10

90% 146 (rounded to 150) Dengue NS1/IgM positive samples: 18 Chikungunya IgM positive samples:18

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Serum reactive for RA factor – low positive and high positive:18 Serum reactive for TPHA/other specific test for syphilis:18 Healthy controls from endemic regions: 78

699 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and 700 specificity; however, a higher sample size is suggested to ensure adequate power of the study 701 in case the kit falls short of claimed performance characteristics. 702

703 Sample panel composition:
704 A. Positive samples: Malaria positive samples should be obtained from health facilities 705 and confirmed using two FDA approved PCR Kits (including Snounou protocol). Once 706 the positive samples are well-characterized with these two PCR assays (100% 707 agreement between results), they should be classified as per their parasite load using 708 ELISA on in-house calibrated equipment. Samples with analyte values satisfying the 709 range of acceptance criteria (as mentioned in this document) should be included in the 710 positive sample panel for the evaluation of malaria RT-PCR kits.
711 Additional analytes (whose cutoff values have not yet been established) may be used 712 for further sample characterization by ELISA. However, this characterization of 713 samples should also be performed on calibrated equipment, leading to their 714 classification as low and high parasitemia samples, which should then be used for 715 performance evaluation of the assay.
716

717 Range of Parasitemia: Panel members should have a low (≤200 parasites per 718 microliter) to high (≥2000 parasites per microliter) range of Plasmodium falciparum, 719 P. vivax, as obtained from ELISA results. Characterized panels must contain equal 720 number of samples of both low and high parasitemia. 721 Consistent ELISA quantification results should be obtained in ≥3 runs of ELISA 722 experiments performed for each of the three antigens (PfHRP2, LDH and aldolase), 723 with the results obtained at the 200 p/µL and the 2,000 p/µL being consistent with each 724 other as well (factor of roughly 10 between results). The limit of detection of Pfhrp2 is 725 5-10 ng/ µL, and Pvldh is 15-45 ng/ µL.
726

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** It should be noted that no such limit of detection is defined for aldolase. Where values/standard 727 reference assay not available, standard procedure on calibrated equipment will be followed for obtaining 728 results. 729 The above mentioned activities should not be performed with spiked/contrived 730 samples. 731 Equal representation of samples positive for all Plasmodium (P.falciparum /P.vivax) 732 species preferred. 733

734 7. Test reproducibility 735 A. Lot-to-lot reproducibility:
736 • Sample size for lot-to-lot reproducibility 737 Three lots of an assay shall be evaluated. The first lot shall be evaluated on the 738 entire panel of samples (statistically significant sample size). For the subsequent 739 two lots, 25 samples should be used for evaluation (15 positive samples including 740 10 weak positive samples and 5 moderate/strong positive samples, and 10 negative 741 samples).
742 743

744

745

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746

747 B. Reader-to-reader reproducibility: 25 samples (15 positive samples including 10 748 weak positive samples and 5 strong/moderate positive samples, and 10 negative 749 samples) need to be tested by at least 2 trained personnel. Agreement should be 100%. 750 C. Machine-to-machine reproducibility: 25 samples (15 positive samples including 10 751 weak positive samples and 5 strong/moderate positive samples, and 10 negative 752 samples) to be tested on two different platforms (e.g.: ABI 7500 and BioRad CFX96). 753 Agreement should be 100%. 754

755 8. Testing Methodology:
756 The reference assay and the index test should be run on the sample panel in parallel. 757

758 9. Interpretation of results:
759 Results should be interpreted as per the IFU of the reference assay and the index test.
760

761 10. Resolution of discrepant results: 762 True positive samples: These are samples positive by both reference assay and index 763 test. 764 True negative samples: These are samples negative by both reference assay and index 765 test. 766 767 index test. 768 769 index test. 770

771 11. Acceptance Criteria: 772 Target Plasmodium species Acceptance criteria Minimum no. of samples needed to achieve at least the performance characteristics outlined in the acceptance criteria

Pf PCR Sensitivity ≥98% Specificity ≥98% Limit of detection: 1 parasite/µl Minimum no. of Positive samples = 80

Minimum no. of Negative samples = 80 Pv PCR Sensitivity ≥95% Specificity ≥98% Limit of detection: 1-2 parasites/µl Minimum no. of Positive samples = 80

Minimum no. of Negative samples = 80 Multiplex PCR - Pf & Pv
For Pf:
• Sensitivity: ≥98%
• Specificity: ≥98% For Pf: Minimum no. of Positive samples = 80

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

• Absolute precision 5%
• 95% CI • Invalid test rate ≤5% • Limit of detection: 1 parasite/µl

For Pv: • Sensitivity: ≥95%
• Specificity: ≥98% • Absolute precision 5%
• 95% CI • Invalid test rate ≤5% • Limit of detection: 1-2 parasites/µl

Minimum no. of Negative samples = 80

For Pv: Minimum no. of Positive samples = 80

Minimum no. of Negative samples = 80

773 Cross-reactivity: nil 774 775

776

777

778 12. Blinding of laboratory staff 779 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should be 780 blinded to the status of the clinical samples. The PI of the evaluation exercise should remain 781 unblinded, i.e., privy to the status of the samples. Another senior laboratory staff selected by 782 the PI may remain unblinded and carry out coding of samples and dispensing them into similar- 783 looking vials to be used for testing, and maintaining the database of results. Staff performing 784 the reference test and the test under evaluation, interpretation of the test result, and entering the 785 results against the coded samples in the database, should remain blinded to the status of samples 786 till the completion of evaluation. The data should be analyzed only by the PI of the evaluating 787 lab. Refer to Fig. 2. 788

789 790

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

791

792

793

794 13. Publication Rights 795 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead 796 author(s). 797

798 After following due procedure as defined in this document, once any kit is found to be 799 Not of Standard Quality, thereafter, no request for repeat testing of the same kit will be 800 acceptable.
801 Any request of re-validation from the same manufacturer for the same test type will only 802 be entertained after a minimum of 3 months and only if a high-level technical summary 803 of modifications or functional improvements to the kit design is submitted, without 804 explicit disclosure of proprietary information. 805 Clinical samples are precious, therefore, repeat evaluation of a kit using the same/ 806 different well-characterized sample panel at a different laboratory may be considered 807 only for kits which claim high performance characteristics (sensitivity and specificity 808 95% and above), but which fail the performance evaluation by a margin of 5%. 809

810

811 VI. References: 812 1. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Do Rosario VE, et al. 813 High sensitivity of detection of human malaria parasites by the use of nested 814

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polymerase chain reaction. Molecular and Biochemical Parasitology. 1993;61:315– 815 20. 816 2. Ramírez AM, Tang THT, Suárez ML, Fernández AÁ, García CM, Hisam S, Rubio 817 JM. Assessment of Commercial Real-Time PCR Assays for Detection of Malaria 818 Infection in a Non-Endemic Setting. Am J Trop Med Hyg. 2021 Oct 819 12;105(6):1732-1737. doi: 10.4269/ajtmh.21-0406. PMID: 34662870; PMCID: 820 PMC8641344. 821 3. Bouzayene, A., Zaffaroullah, R., Bailly, J. et al. Evaluation of two commercial kits 822 and two laboratory-developed qPCR assays compared to LAMP for molecular 823 diagnosis of malaria. Malar J 21, 204 (2022). https://doi.org/10.1186/s12936-022- 824 04219-1 825

826 4. Aschar M, Sanchez MCA, Costa-Nascimento MJ, Farinas MLRN, Hristov AD, 827 Lima GFMC, Inoue J, Levi JE, Di Santi SM. Ultrasensitive molecular tests 828 for Plasmodium detection: applicability in control and elimination programs and 829 reference laboratories. Rev Panam Salud Publica. 2022 Mar 28;46:e11. doi: 830 10.26633/RPSP.2022.11. PMID: 35355692; PMCID: PMC8959250. 831

832 VII. Performance evaluation report format 833

834

835

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845

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848

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REPORT FORMAT 853 Name of the Laboratory 854 Name of the Institute, (with station) 855 Certificate of Analysis 856 File No.: _________________________________________ 857 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Sample type Positive samples (provide details: strong, moderate, weak)

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Negative samples (provide details, including cross reactivity panel)

858

859 Results: 860

861

Reference assay ……….……………… (name)

Positive Negative Total Name of malaria real time PCR kit Positive

Negative

Total

862

863

Estimate (%) 95% CI Sensitivity

Specificity

864 ● Details of cross reactivity with other agents:
865 ● Conclusions: 866 o Sensitivity, specificity 867 o Performance: Satisfactory / Not Satisfactory 868 (Sensitivity and specificity have been assessed in controlled lab setting on ……. samples only, 869 using kits provided by the manufacturer from the batch mentioned above. Results should not 870 be extrapolated for any other sample type.) 871 Disclaimers 872 873 874

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by …………… 875 (Supplied by ……….) 876

877 878 879

880 Signature of Director/ Director-In-charge ……………………

Seal 881 ……………………………………… 882 ****End of the Report 883

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Field evaluation protocol for combo Malaria Rapid Diagnostic Test (RDT) kits 906 (detecting P vivax and P falciparum) 907 I. Background:
908 CDSCO/ICMR, New Delhi, have aimed to facilitate the availability of Quality-Assured 909 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish the 910 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 911 evaluation is to independently verify the manufacturer’s claim regarding IVD kit performance. 912 II. Purpose: 913 To evaluate the performance characteristics of Malaria RDT kits (detecting P. vivax and/or P. 914 falciparum) in the diagnosis of Malaria parasite infection in individuals with unknown disease 915 status. 916 III. Requirements:
917

Supply of kits under evaluation (with batch no. and lot no. Manufacturing and Expiry 918 dates other required details). If the kit to be evaluated works in a closed system format, 919 the manufacturer needs to supply the required equipment. 920
Evaluation sites/laboratories (With required equipment) 921
Reference test kits 922
Laboratory supplies
923 IV. Ethical approval:
924 The study will be initiated after approval from the institutional human ethics committee.
925 V. Procedure: 926
Study design/type: Cross-sectional study 927 928 Identified IVD kit evaluation laboratories should establish their proficiency through
929 A. Laboratory accreditation: Accreditation for at least one of the Quality management 930 systems (accreditation for Testing Lab / Calibration Lab (ISO: 17025), Medical Lab (ISO: 931 15189), PT provider (ISO: 17043) or CDSCO approved Reference laboratory. 932 It is recommended that malaria Medical Device Testing Labs (MDTLs) participate in 933 Quality Control exercises such as EQAP (External Quality Assurance Programme). 934

935 B. Staff training: All the staff involved in IVD kit evaluation should undergo hands on 936 training and competency testing on the following at referral level malaria labs before 937 initiation of MDTL activity:
938 ⮚ Preparation and characterization of evaluation panel for the respective IVD kit. 939 ⮚ Management of RDT kits (specific for Plasmodium falciparum / Plasmodium vivax) 940 received for performance evaluation (Verification/Storage/Unpacking etc.). 941

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

⮚ Perform tests interpretation and documentation of results, and reporting. 942 ⮚ Data management and safety and confidentiality. 943 3. Sample size for performance evaluation: 944 Sample sizes of positive and negative samples against different values of sensitivity and 945 specificity are provided in Tables 1 and 2. Sample sizes have been calculated assuming 946 95% level of significance, an absolute precision of 5%, and invalid test rate 5%. It is further 947 assumed that at least 5% of the individuals attending the health care facilities for acute 948 febrile illness and suspected for Malaria will be positive for Malaria (P. vivax and P. 949 falciparum). Appropriate sample size has to be chosen from the tables according to the 950 values of sensitivity and specificity being claimed by the manufacturer. If a claimed 951 sensitivity/specificity is not present in the table, the manufacturer needs to consider the 952 sample size associated with the largest sensitivity/specificity provided in the table that is 953 smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ 954 specificity available in the table). For example, if a manufacturer claims a sensitivity of 955 93%, they are required to use a sample size mentioned against 90% sensitivity. Similarly, 956 a claim of 87% specificity would require usage of the sample size outlined for 85% 957 specificity. Sample sizes are calculated using the following formulae and assumption of 958 5% for prevalence of the disease: 959

960 𝑑2 x (1 −IR) x P 961 𝑑2 x (1 −IR) x P 962

963

964 · n (se) is the minimum number of individuals to be enrolled to obtain the 965 requisite number of positive samples. 966 · n (sp) is the minimum number of individuals to be enrolled to obtain the 967 requisite number of negative samples. 968 · Z2 is the critical value from the standard normal distribution 969 corresponding to the desired confidence level (95% CI corresponds to Z2 970 =1.96). 971 972 973 974 975

P is prevalence of the disease 976

977 Sample size has to be calculated based on both the sensitivity and the specificity. The 978 final sample size will be the maximum of the two. For example, at 95% sensitivity and 979

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

95% specificity, the sample size required will be 1600 (maximum of 1600 and 84). 980 Please note that since the prevalence is low, the final sample size is generally expected 981 to be governed by the assumed sensitivity.
982

983 Table 1. Sample sizes for different values of species-specific sensitivity being claimed
984 Sensitivity Minimum no. of positive samples required (rounded figure) # Minimum number of individuals to be enrolled in the study to obtain requisite number of positive samples 99% 20 400 95% 80 1600 90% 150 3000 85% 210 4200 80% 260 5200 75% 305 6100 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and specificity; however, a higher sample size is suggested to ensure adequate power of the study in case the kit falls short of claimed performance characteristics. Samples will be collected from individuals attending the health care facilities (tertiary care centers and their linked hospitals, private clinics, field practice areas etc.) for acute febrile illness in highly endemic areas.

The disease status of these cases will be unknown.

985 Table 2. Sample sizes for different values of species-specific specificity being claimed 986 Specificity No. of negative samples required (rounded figure) Minimum number of individuals to be enrolled to obtain requisite number of negative samples 99% 20 21 95% 80 84 90% 150 158 85% 210 221 80% 260 274 75% 305 321 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and specificity; however, a higher sample size is suggested to ensure adequate power of the study in case the kit falls short of claimed performance characteristics. Samples will be collected from individuals attending the health care facilities (tertiary care centers and their linked hospitals, private clinics, field practice areas etc.) for acute febrile illness in highly endemic areas.

987

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Since a large number of febrile cases have to be enrolled to obtain the requisite number of 988 malaria positive samples, enrolling the number of cases mentioned in Table 1 will be sufficient 989 to obtain the requisite number of negative samples. 990 4. Inclusion criteria: 991 Individuals with the following clinical features may be enrolled in the study 992 Fever and any 2 of the following: 993 o Chills, sweating, headache, tiredness, nausea and vomiting, jaundice, splenomegaly
994 5. Exclusion criteria 995 • Individuals not satisfying inclusion criteria 996 • Individuals with already known positive history for other pathogens 997 6. Reference assay:
998 WHO Pre-Qualified/ US FDA/ ATAGI Australia/ PMDA Japan approved Malaria PCR assay/ 999 Snounou protocol should be used as reference assay. 1000 7. Study implementation: 1001 The patients displaying Malaria like illness will be recruited into the study and five ml of whole 1002 blood will be collected in EDTA tubes. The whole blood sample will be subjected to the 1003 reference and the index test. 1004 The disease status of the enrolled cases will be unknown. 1005 8. Evaluation method: 1006 The index test and the reference tests should be run simultaneously on the sample panel, and 1007 results should be recorded.
1008

1009 9. Interpretation of results:
1010 Reference test and index test results will be interpreted as per kit IFU.
1011 10. Positive samples: 1012 Samples positive by the reference assay will be considered as true positive samples.
1013 11. Negative samples:
1014 Samples negative by the reference assay will be considered as true negative samples. 1015 False positive samples: These are samples negative by reference assay and positive by index 1016 test. 1017 False negative samples: These are samples positive by reference assay and negative by index 1018 test. 1019

1020 A. Cross reactivity: 1021

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The RDT kit should have been evaluated against the following cross reactivity panel during 1022 the analytical performance evaluation: 1023

1024 • Dengue NS1 positive samples (n=10 samples) 1025 • Chikungunya PCR positive samples (n=10 samples) 1026 • Healthy controls from endemic regions (n= 40 samples) 1027 • Serum reactive for RA factor – low positive and high positive (n=15 1028 samples) 1029 • Serum reactive for TPHA/other specific test for syphilis (n= 10 samples) 1030 12. Statistical analysis:
1031 Sensitivity and specificity will be calculated. 1032 Interim analysis of data shall be conducted on completing evaluation of 25%, 50% and 75% of 1033 samples. If, at any point, the performance of the assay is found to be not satisfactory, the assay 1034 shall not be evaluated further. Evaluation fee shall be charged accordingly. 1035 13. Test reproducibility 1036 A. Sample size for lot-to-lot reproducibility 1037 Three lots of an assay shall be evaluated. The first lot shall be evaluated on the entire panel 1038 of samples (statistically significant sample size). For the subsequent two lots, 25 samples 1039 should be used for evaluation (15 positive samples including 10 weak positive samples and 1040 5 moderate/strong positive samples, and 10 negative samples).
1041 1042

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1043

1044 B. Reader-to-reader reproducibility: 25 samples (15 positive samples including 10 1045 weak positive samples and 5 strong/moderate positive samples, and 10 negative 1046 samples) need to be tested by at least 2 trained personnel. Agreement should be 100%. 1047

1048 14. Resolution of discrepant results: 1049 True positive samples: These are samples positive by both reference assay and index 1050 test. 1051 True negative samples: These are samples negative by both reference assay and index 1052 test. 1053 1054 index test. 1055 1056 index test. 1057

1058 15. Blinding of laboratory staff 1059 To ensure rigor of the evaluation process, laboratory staff performing the evaluation 1060 should be blinded to the results of the reference assay. The PI of the evaluation exercise 1061 should remain unblinded, i.e., privy to the results of the reference test. Another senior 1062 laboratory staff selected by the PI may remain unblinded for overseeing the activity and 1063

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

maintaining the database of results.. The data should be analyzed only by the PI of the 1064 evaluating lab.
1065 16. Acceptance criteria: 1066 Expected sensitivity: ≥75% for P. vivax and ≥95% for P. falciparum 1067 Expected specificity: ≥90% for P. vivax and ≥95% for P. falciparum 1068 1069 1070

1071 To achieve at least the performance characteristics outlined in the acceptance criteria 1072 for P vivax, ≥6100 individuals satisfying the case definition need to be enrolled to 1073 obtain the requisite number of positive samples. This sample size is sufficient for 1074 requisite number of negative samples. 1075 To achieve at least the performance characteristics outlined in the acceptance criteria 1076 for P falciparum, ≥1600 individuals satisfying the case definition need to be enrolled 1077 to obtain the requisite number of positive samples. This sample size is sufficient for 1078 requisite number of negative samples. 1079

1080 Recruitment should be terminated once the desired number of positive cases is enrolled 1081 and tested. 1082

1083 17. Publication Rights 1084 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead 1085 author(s). 1086 After following due procedure as defined in this document, once any kit is found to be 1087 Not of Standard Quality, thereafter, no request for repeat testing of the same kit will be 1088 acceptable.
1089 Any request of re-validation from the same manufacturer for the same test type will only 1090 be entertained after a minimum of 3 months and only if a high-level technical summary 1091 of modifications or functional improvements to the kit design is submitted, without 1092 explicit disclosure of proprietary information. 1093 Clinical samples are precious, therefore, repeat evaluation of a kit using the same/ 1094 different well-characterized sample panel at a different laboratory may be considered 1095 only for kits which claim high performance characteristics (sensitivity and specificity 1096 95% and above), but which fail the performance evaluation by a margin of 5%. 1097

1098 VI. References: 1099 1. Ministry of Health and Family Welfare. Guidelines for Bivalent RDT. Available at: 1100 guidelines-for-bivalent-rdt.pdf (mohfw.gov.in) 1101 2. World Health Organization. Malaria Rapid Diagnostic Test Performance - Results 1102 of WHO product testing of malaria RDTs: round 8 (2016–2018): Available at: 1103 https://iris.who.int/bitstream/handle/10665/276190/9789241514965- 1104 eng.pdf?sequence=1
1105 3. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Do Rosario VE, et al. 1106 High sensitivity of detection of human malaria parasites by the use of nested 1107

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polymerase chain reaction. Molecular and Biochemical Parasitology. 1993;61:315– 1108 20. 1109 4. Integrated Disease Surveillance Project Training Manual For State & District 1110 Surveillance Officers - Case Definitions Of Diseases & Syndromes Under 1111 Surveillance (Module-5). Available at: 1112 https://idsp.mohfw.gov.in/WriteReadData/OldSite/2WkDSOSept08/Resources_fil 1113 es/DistrictSurvMan/Module5.pdf [Accessed on 25th June 2024] 1114 5. CDC. National Notifiable Diseases Surveillance System (NNDSS). Malaria 1115 (Plasmodium spp.) 2014 Case Definition. Available at: 1116 https://ndc.services.cdc.gov/case-definitions/malaria-2014/ [Accessed on 28th June, 1117 2024] 1118 6. Kannambath R, Rajkumari N, Sivaradjy M. Prevalence of malaria: A 7-year trend 1119 analysis from a tertiary care center, Puducherry. Trop Parasitol. 2023 Jan- 1120 Jun;13(1):28-33. doi: 10.4103/tp.tp_41_22. Epub 2023 May 19. PMID: 37415756; 1121 PMCID: PMC10321582. 1122 VII. Performance evaluation report format 1123

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REPORT FORMAT 1144 Name of the Laboratory 1145 Name of the Institute, (with station) 1146 Certificate of Analysis 1147 File No.: _________________________________________ 1148 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

Valid Up to: Application No. Sample Panel Sample type Positive samples (provide details: strong, moderate, weak)

ICMR-CDSCO/IVD/GD/PROTOCOLS/06/2025

Negative samples (provide details, including cross reactivity panel)

1149

1150 Results: 1151

1152

Reference assay ……….……………… (name)

Positive Negative Total Name of index malaria RDT Positive

Negative

Total

1153

1154

Estimate (%) 95% CI Sensitivity

Specificity

1155 ● Details of cross reactivity with other agents:
1156 ● Conclusions: 1157 o Sensitivity, specificity 1158 o Performance: Satisfactory / Not Satisfactory 1159 (Sensitivity and specificity have been assessed in field/controlled lab setting on……. samples 1160 only, using kits provided by the manufacturer from the batch mentioned above. Results 1161 should not be extrapolated for any other sample type.) 1162 Disclaimers 1163 1164 1165

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Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by 1166 …………… (Supplied by ……….) 1167

1168 1169 1170

1171 Signature of Director/ Director-In-charge …………………… Seal 1172 ……………………………………… 1173 ****End of the Report 1174

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Information on Operational and Test Performance Characteristics Required from 1197 Manufacturers for Malaria IVD 1198 The manufacturer should provide the following details about the IVD: 1199

Instructions for Use 1200
Scope of the IVD: to diagnose Malaria (Pf and/or Pv) 1201
Intended Use Statement 1202
Principle of the assay 1203
Intended testing population (cases of acute febrile illness/suspected cases of Malaria) 1204
Intended user(laboratory professional and/or health care worker at point-of-care) 1205
Detailed test protocol 1206
Lot/batch No. 1207
Date of manufacture 1208
Date of Expiry 1209
Information on operational Characteristics 1210 i. Configuration of the kit/device 1211 ii. Requirement of any additional equipment, device 1212 iii. Requirement of any additional reagents 1213 iv. Operation conditions 1214 v. Storage and stability before and after opening 1215 vi. Internal control provided or not 1216 vii. Quality control and batch testing data 1217 viii. Biosafety aspects- waste disposal requirements 1218
Information on Test Performance Characteristics 1219 i. Type of sample-serum/plasma/whole blood/other specimen (specify) 1220 ii. Volume of sample 1221 iii. Sample rejection criteria (if any) 1222 iv. Any additional sample processing required 1223 v. Any additional device/consumable like sample transfer device, pipette, tube, etc 1224 required 1225 vi. Name of analyte to be detected 1226 vii. Pathogens targeted by the kit 1227

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viii. Time taken for testing 1228 ix. Time for result reading and interpretation 1229 x. Manual or automated(equipment)reading 1230 xi. Limit of detection 1231 xii. Diagnostic sensitivity 1232 xiii. Diagnostic specificity 1233 xiv. Stability and reproducibility (including data) 1234 xv. Training required for testing (if any) 1235 xvi. If yes, duration 1236 xvii. Details of Cut-off and /or Equivocal Zone for interpretation of test 1237 xviii. Details of cross reactivity, if any 1238 xix. Interpretation of invalid and indeterminate results to be provided 1239 xx. It is recommended to provide data demonstrating the precision 1240 xxi. Limit of detection 1241

1242 *Please mention “Not applicable” against sections not pertaining to the kit. 1243

1244

1245 ****End of the Document 1246

1247

1248

1 STANDARD PERFORMANCE 2 EVALUATION PROTOCOL
3 DRAFT FOR STAKEHOLDER COMMENTS 4

5 NIPAH VIRUS REAL TIME PCR KIT 6 ICMR-CDSCO/IVD/GD/PROTOCOLS/08/2025 7

9 AUGUST, 2025 New Delhi, India

Nipah Virus Real Time PCR Kit Performance Evaluation Protocol ICMR-CDSCO/IVD/GD/PROTOCOLS/08/2025 Page 1 of 15

Table of Contents 10 S.N. Topic Page Number 1.
Performance evaluation protocol for Nipah virus real-time PCR kits 2 2.
Performance Evaluation Report Format 11 3.
Information on Operational and Test Performance Characteristics Required from Manufacturers 13

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Performance evaluation protocol for Nipah virus real-time PCR kit 36 I. Background: 37 CDSCO and ICMR, New Delhi, have aimed at facilitating the availability of Quality-Assured 38 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish the 39 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 40 evaluation is to independently verify the manufacturer’s claim regarding in-vitro diagnostic kit 41 (IVD) performance. 42 This recommendation focuses on the laboratory performance evaluation of Nipah virus real time 43 PCR kit. All clinical samples tested in the study should be evaluated in accordance with the 44 candidate test’s instructions for use.
45 II. Purpose: 46 To evaluate the performance characteristics of Nipah virus real-time PCR kits in the diagnosis of 47 Nipah virus infection/ disease using irreversibly de-identified leftover archived/ spiked clinical 48 samples. 49 III. Requirements:
50

Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 51 the kit to be evaluated works in a closed system format, the manufacturer needs to supply 52 the required equipment. 53
Evaluation sites/laboratories (With required equipment) 54
Reference test kits 55
Characterised Evaluation panel 56
Laboratory supplies
57 IV. Ethical approvals:
58 Performance evaluation activities using irreversibly de-identified leftover clinical samples are 59 exempt from ethics approval as per ICMR’s Guidance on Ethical Requirements for Laboratory 60 Validation Testing, 2024.
61 Investigators are required to submit a self-declaration form, as outlined in the ICMR 62 guidelines, to the institutional authorities and ethics committee for information. 63 V. Procedure: 64
Study design/type: Diagnostic accuracy study using spiked/clinical samples (human 65 specimens). 66 67

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Identified IVD kit evaluation laboratories should be well-equipped and establish their 68 proficiency through ALL of the following: 69

70 A. Availability of BSL-4 facility for handling of Nipah virus positive specimens 71 B. Accreditation for at least one Quality management system for at least one respiratory viral 72 pathogen molecular testing (accreditation for Testing Lab / Calibration Lab as per ISO/IES 73 17025, Medical Lab as per ISO 15189, PT provider as per ISO/IEC 17043), or CDSCO 74 approved Reference laboratory.
75 C. Staff training: All the staff involved in Nipah virus IVD evaluation should undergo hands on 76 training and competency testing on following
77 ⮚ BSL-4 practices
78 ⮚ Nipah virus culture and handling 79 ⮚ Preparation & characterization of reference sample panel 80 ⮚ Handling of Nipah virus RT-PCR kits received for performance evaluation 81 (Verification/Storage/Unpacking etc). 82 ⮚ Testing, interpreting, recording of results & reporting 83 ⮚ Data handling, data safety & confidentiality 84 3. Preparation of Nipah virus RNA evaluation panel 85 This is a zoonotic disease, and well characterised Nipah virus positive human samples is a critical 86 requirement for evaluation of RT-PCR IVD kits. A statistically significant number of clinical 87 samples should be used for the evaluation. 88 4. RNA extraction 89 RNA extraction should be performed as per manufacturer’s instruction for reference assay as well 90 as the assay under evaluation. If any extraction system is specified in the IFU, that shall be used 91 for the test and shall be provided by the manufacturer.
92 5. Real-Time PCR System 93 PCR shall be performed using IVD-approved machines. If any equipment(s) is specified in the 94 IFU, that shall be used for the test and shall be provided by the manufacturer.
95 Real-time closed systems/devices awaiting evaluation should be provided by the manufacturer 96 along with all necessary components, supplies and reagents. 97 6. Internal control/Extraction control 98 Assays must have an internal control (housekeeping gene), with or without an extraction control 99 (RNA added before extraction to a sample).
100

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Reference assay:
101 The Nipah virus Real Time PCR Assay developed by ICMR-NIV Pune, or a WHO Pre-Qualified/ 102 US FDA/ ATAGI Australia/ PMDA Japan approved real time PCR assay should be used as the 103 Reference Standard.
104 All positive samples should be confirmed positive by the reference assay. 105 All negative samples should be confirmed negative by the reference assay. 106

107 8. Sample size for performance evaluation: Sample size is calculated assuming 95% 108 sensitivity and specificity of the index test, 95% confidence level, absolute precision of 5% and 109 ≤5% invalid test rate. A minimum of 77 (rounded to 80) positive clinical samples and a minimum 110 of 77 (rounded to 80) negative clinical samples are required. Sample sizes are calculated using the 111 formulae: 112

113 𝑑2 x (1 −IR)
114 𝑑2 x (1 −IR)
115

116

117 118 119 120 121 122 123 124 125

126 Nipah virus is detectable from throat swab, urine, CSF. The assay should be validated with positive 127 clinical/spiked samples, and negative samples for all the formats claimed by the manufacturer. 128 However, if a particular sample matrix is used to evaluate the assay (as opposed to all the sample 129 types claimed by the manufacturer), the performance evaluation report should clearly mention the 130 performance characteristics of the assay against the sample type used for validation. There should 131 be no ambiguity about the sample type used for assay validation. 132

133

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134

135 9. Sample panel composition: 136 A. Human samples 137 A.1 Positive samples (Minimum n=80 for each sample type): Clinical/ Spiked samples 138 positive by the reference real-time PCR assay
139 A.1.1 Strong positive (Ct value <25) = 24 samples
140 A.1.2. Moderate positive (Ct value between 25-30) = 28 samples 141 A.1.3 Weak positive (Ct value >30 to 34) = 28 samples
142 The sample type should be as per the index test IFU. If an assay claims to detect Nipah 143 virus RNA in several sample types, attempt should be made to use 80 positive samples 144 across each sample type, or at least the sample types available with the evaluating lab. This 145 relaxation is provided since clinical samples are scarce and obtained only during outbreaks 146 occurring every few years in India, which necessitates using spiked clinical samples. The 147 latter is difficult since Nipah virus is a BSL-4 level pathogen and its handling requires 148 sophisticated laboratory setup and trained manpower. 149 In case the requisite number of specimens for a particular sample type are not available and 150 a smaller number of samples are used for performance evaluation (i.e., sample size 151 calculated assuming higher performance characteristics), it is necessary to ensure that the 152 study has adequate power for acceptance of the evaluation results in case the assay falls 153 short of the assumed performance characteristics. 154 Note: 155 If clinical samples positive for Nipah virus are not available, tissue culture fluid (Heat-inactivated) from reference 156 laboratories can be used, spiked in serum/urine/Throat swab samples to obtain the panel with Ct value <25, 25-30 and 157

35 and tested by the reference assay, and the positive samples can be used for evaluation.
158 Confirmed negative samples would be used for spiking with Nipah virus.isolate.
159

160 A.2 Negative samples (number of samples will depend on sample type): All negative 161 samples should be negative by reference real-time PCR assay. Distribution of the negative 162 samples should be as follows 163 Categories of samples as per the sample type Sample type
NP/TS (Minimum n= 80) Serum (Minimum n= 80) Urine (Minimum n=80)

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A.2.1 Samples from cases having similar illness/ spiked samples which are RT- PCR positive for common pathogens but negative for Nipah virus Samples from individuals presenting with ARI/ILI/SARI (n=45):

5 positive clinical/ spiked samples from each of the following diseases:

Influenza A virus @
Influenza B virus @
SARS-CoV-2 @
RSV A/B @
HPIV @
HMPV @
Adenovirus @
Seasonal Coronaviruses *
Rhinovirus/Enterovirus*

Cross reactivity panel is arranged in descending order of priority. The pathogens marked @ are essentially to be tested. It is recommended to test for all pathogens listed in the cross reactivity panel. However, if there is an acute shortfall or non- availability of clinical samples, one may consider reducing only the pathogens of lower priority marked by * , while ensuring that the actual numbers of cross reactive sample panel remain the same by compensating with the available “essentially to be tested” samples. Samples from cases of AES (n=35):

5 positive clinical/ spiked samples from each of the following diseases:

Japanese Encephalitis @
Dengue @
HSV @
VZV @
West Nile Virus *
Chandipura virus *
Rabies virus *

Cross reactivity panel is arranged in descending order of priority. The pathogens marked @ are essentially to be tested. It is recommended to test for all pathogens listed in the cross reactivity panel. However, if there is an acute shortfall or non-availability of clinical samples, one may consider reducing only the pathogens of lower priority marked by * , while ensuring that the actual numbers of cross reactive sample panel remain the same by compensating with the available “essentially to be tested” samples. 5 positive clinical/ spiked samples from each of the following diseases, presenting with respiratory and/or encephalitis symptoms (n=20):

Measles
Rubella
Mumps
SARS-CoV- 2

A.2.2 Samples from cases with acute respiratory disease/ acute encephalitis/ acute febrile illness and RT- PCR negative for the above- mentioned pathogens and Nipah virus 25 35 40

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A.2.3 Healthy/ asymptomatic cases

from endemic regions negative for Nipah virus 10
10 20 Serum/ throat swab/ urine samples collected from the same case may be used for evaluation. 10. Evaluation method: 164 The index test and the reference tests should be run simultaneously on the sample panel, 165 and results should be recorded.
166

167 11. Interpretation of results:
168 Reference test and index test results will be interpreted as per kit IFU.
169

170

171 12. Resolution of discrepant results: 172 True positive samples: These are samples positive by reference assay and index test. 173 True negative samples: These are samples negative by reference assay and index test. 174 175 index test. 176 177 index test. 178

179 13. Test reproducibility 180 A. Sample size for lot-to-lot reproducibility 181 Three lots of an assay shall be evaluated. Sample size for lot-to-lot reproducibility should be 182 as follows:
183 184 and negative samples as calculated in the protocol.
185 • Second lot of the assay: should be tested on 25 samples (15 positive samples 186 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 187 samples).
188 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 189 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 190

191 192

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193

194 14. Blinding of laboratory staff 195 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should be 196 blinded to the status of the clinical samples. The PI of the evaluation exercise should remain 197 unblinded, i.e., privy to the status of the samples. Another senior laboratory staff selected by the 198 PI may remain unblinded and carry out coding of samples and dispensing them into similar- 199 looking vials to be used for testing, and maintaining the database of results. Staff performing the 200 reference test and the test under evaluation, interpretation of the test result, and entering the results 201 against the coded samples in the database, should remain blinded to the status of samples till the 202 completion of evaluation. The data should be analyzed only by the PI of the evaluating lab. Refer 203 to Fig. 2. 204

205 206

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207

208 15. Acceptance Criteria 209 Expected sensitivity: ≥95% 210 Expected specificity: ≥98% 211 Cross reactivity with other viruses as outlined in the negative sample panel: Nil 212 213

214 16. Publication Rights: 215 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead author(s). 216

217 218 219 acceptable.
220 221 222 223 224

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225 226 227 228

229 VI. References: 230

Yadav PD, Majumdar T, Gupta N, Kumar MA, Shete A, Pardeshi P, Sultana S, Sahay RR, Manoj 231 MN, Patil S, Floura S, Gangakhedkar R, Mourya DT. Standardization & validation of Truenat™ 232 point-of-care test for rapid diagnosis of Nipah. Indian J Med Res. 2021 Apr;154(4):645-649. doi: 233 10.4103/ijmr.IJMR_4717_20. PMID: 34854433; PMCID: PMC9205002. 234
World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 235 Diagnostic Assessment TGS-3.

Available at: 236 https://iris.who.int/bitstream/handle/10665/258985/WHO-EMP-RHT-PQT-TGS3-2017.03- 237 eng.pdf;sequence=1
238

239 VII. Performance evaluation report format 240

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PERFORMANCE EVALUATION REPORT FOR NIPAH VIRUS REAL-TIME PCR 257 KITS 258 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

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

259 Results 260

Reference assay ……….……………… (name)

Positive Negative Total Name of Nipah virus real-time PCR Positive

Negative

Total

261

Estimate (%) 95% CI Sensitivity

Specificity

262 ● Details of cross reactivity with other Paramyxoviruses: 263 ● Conclusions: 264

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o Sensitivity, specificity 265 o Cross reactivity 266 o Invalid test rate 267 o Performance: Satisfactory / Not satisfactory 268 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 269 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 270 Disclaimers 271 272 273 Note: This report is exclusively for Nipah virus………….. Kit (Lot No……) manufactured by …………… 274 (supplied by ……….) 275 276 277 Signature of Director/ Director-In-charge …………………… Seal …………………………. 278 ****End of the Report 279

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Annexure-1: Information on Operational and Test Performance Characteristics Required 296 from Manufacturers 297 The manufacturer should provide the following details about the IVD: 298

Instructions for Use 299
Scope of the IVD: to diagnose Nipah virus 300
Intended Use Statement 301
Principle of the assay 302
Intended testing population (cases of AES/ARI/SARI) 303
Intended user (laboratory professional and/or health care worker at point-of-care) 304
Lot/batch No. 305
Date of manufacture 306
Date of Expiry 307
Information on operational Characteristics 308 i. Configuration of the kit/device 309 ii. Requirement of any additional equipment, device 310 iii. Requirement of any additional reagents 311 iv. Operation conditions 312 v. Storage and stability before and after opening 313 vi. Internal control provided or not 314 vii. Quality control and batch testing data 315 viii. Biosafety aspects- waste disposal requirements 316
Information on Test Performance Characteristics 317 i. Type of sample- Nasopharyngeal swab/Throat swab/ CSF/Serum / Other specimen 318 ii. Volume of sample 319 iii. Any specific sample NOT to be tested 320 iv. Any additional sample processing required 321 v. Any additional device/consumable like sample transfer device, pipette, tube, etc required 322

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vi. Name of analyte to be detected 323 vii. Pathogen(s) targeted by the kit 324 viii. Time taken for testing 325 ix. Time for result reading and interpretation 326 x. Manual or automated(equipment)reading 327 xi. Limit of detection 328 xii. Diagnostic sensitivity 329 xiii. Diagnostic specificity 330 xiv. Stability and reproducibility 331 xv. Training required for testing 332 xvi. If yes, duration 333 xvii. Details of Cut-off and /or Equivocal Zone for interpretation of test 334 xviii. Interpretation of invalid and indeterminate results to be provided 335 xix. It is recommended to provide data demonstrating the precision 336

337 *Please mention “Not applicable” against sections not pertaining to the kit. 338

339

340 ****End of the Document 341

1 STANDARD PERFORMANCE 2 EVALUATION PROTOCOL
3 DRAFT FOR STAKEHOLDER COMMENTS 4

5 CHANDIPURA VIRUS REAL TIME PCR 6 KIT 7 ICMR-CDSCO/IVD/GD/PROTOCOLS/07/2025 8

10 AUGUST, 2025 New Delhi, India

Chandipura Virus Real Time PCR Kit Performance Evaluation Protocol ICMR-CDSCO/IVD/GD/PROTOCOLS/07/2025 Page 1 of 15

Table of Contents 11 S.N. Topic Page Number 1.
Performance evaluation protocol for Chandipura virus real-time PCR kits 2 2.
Performance Evaluation Report Format 11 3.
Information on Operational and Test Performance Characteristics Required from Manufacturers 13

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Performance evaluation protocol for Chandipura virus real-time PCR kits 37 I. Background
38 CDSCO and ICMR, New Delhi, have aimed at facilitating the availability of Quality-Assured 39 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish the 40 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 41 evaluation is to independently verify the manufacturer’s claim regarding in-vitro diagnostic kit 42 (IVD) performance. 43 This recommendation focuses on the laboratory performance evaluation of Chandipura virus 44 (CHPV) virus real time PCR kit. All clinical samples tested in the study should be evaluated in 45 accordance with the candidate test’s instructions for use.
46 II. Purpose: 47 To evaluate the performance characteristics of CHPV real-time PCR kits in the diagnosis of CHPV 48 infection/ disease using irreversibly de-identified leftover archived/ spiked clinical samples. 49

50 III. Requirements:
51

Kits Under Evaluation: Include detailed information such as batch number, lot number, expiry 52 date, and other relevant specifications. For kits designed to operate within a closed system, 53 manufacturers must provide the necessary equipment and consumables for testing. 54
Evaluation Sites/Laboratories: Identify laboratories equipped with the required instruments 55 and infrastructure to conduct the evaluation. 56
Reference Test Kits: Use reference kits or in-house kits developed by the reference laboratory, 57 which have been validated to demonstrate satisfactory performance. 58
Evaluation Panel: Prepare a panel of well-characterised clinical samples from confirmed cases 59 or spiked samples for a comprehensive evaluation. 60
Laboratory Supplies: Ensure all necessary laboratory materials and supplies are available for 61 the evaluation process. 62

63 IV. Ethical Approvals:
64 Performance evaluation activities using irreversibly de-identified clinical samples are exempt from 65 ethics approval as per ICMR’s Guidance on Ethical Requirements for Laboratory Validation 66 Testing, 2024.
67 Investigators are required to submit a self-declaration form, as outlined in the ICMR guidelines, 68 to the institutional authorities and ethics committee for information. 69

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70 V. Procedure:
71

Study design/type: Diagnostic accuracy study using irreversibly de-identified archived 72 clinical/spiked samples
73 74 Identified IVD kit evaluation laboratories should establish their proficiency through the following: 75 A) Accreditation for at least one of the Quality management systems, such as 76 • Testing Laboratory or Calibration Laboratory (ISO/IEC 17025) 77 • Medical Laboratory (ISO 15189) 78 • Proficiency Testing Provider (ISO/IEC 17043) 79 OR 80 • CDSCO-approved reference laboratory 81 B) Staff training: All staff involved in IVD kit evaluation process should undergo hands on training 82 and competency assessment in the following areas:
83 • Preparation and characterization of kit evaluation panel
84 • Handling of Chandipura real-time PCR kits received for performance evaluation 85 (verification/storage/unpacking etc.).
86 • Testing procedures, interpretation and recording of results, and reporting
87 • Data handling, data safety & confidentiality
88
Preparation of Chandipura RNA evaluation panel:
89 A well characterised panel of CHPV positive clinical samples is a critical requirement for 90 evaluation of these RT-PCR IVD kits. A statistically significant number of clinical samples should 91 be used for the evaluation. 92 The sample type for CHPV detection is Cerebrospinal fluid (CSF) and serum. If a kit claims to 93 detect CHPV in both sample types, attempt should be made to evaluate the assay across both serum 94 and CSF using statistically significant sample size for each sample type. In case all the sample 95 types mentioned in the IFU are not available with the lab, the performance evaluation report should 96 clearly mention the sample type against which the kit is evaluated, ensuring statistical rigor. There 97 should be no ambiguity about the type of sample used for evaluation.
98
RNA extraction:
99 RNA extraction should be performed as per manufacturer’s instruction for reference assay as well 100 as the assay under evaluation. If the manufacturer of the index test recommends a specific RNA 101

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extraction kit, it needs to be provided by the manufacturer if the evaluation lab is unable to procure 102 the same.
103 5. Real-time PCR system: 104 PCR should be performed using IVD-approved machines. If any equipment(s) is specified in the 105 IFU of the index test, it should be used for the evaluation, and it should be provided by the 106 manufacturer if not available within the lab’s IVD evaluation scope.
107 Real-time closed systems/devices awaiting evaluation should be provided by the manufacturer 108 along with all necessary components, supplies and reagents. 109 6. Internal Control/Extraction Control: 110 The index test must have an internal control (housekeeping gene), with or without an extraction 111 control (RNA added before extraction to a sample). 112 7. Reference assay: 113 A WHO Pre-Qualified/ US FDA/ ATAGI Australia/ PMDA Japan approved real time CHPV PCR 114 assay/ ICMR-National Institute of Virology, Pune developed protocol for detection of Chandipura 115 virus RNA will serve as the reference assay.
116 All positive samples should be confirmed positive by the reference assay. 117 All negative samples should be confirmed negative by the reference assay and CHPV IgM. 118 8. Sample size for performance evaluation: 119

Sample size is calculated assuming 95% sensitivity and specificity of the index test, 95% 120 confidence level, absolute precision of 5% and ≤5% invalid test rate. A minimum of 77 (rounded 121 to 80) positive clinical samples and a minimum of 77 (rounded to 80) negative clinical samples for 122 each sample type are required for performance evaluation. Sample sizes are calculated using the 123 formulae: 124

125 𝑑2 x (1 −IR)
126 𝑑2 x (1 −IR)
127

128

129 130 131 132 133

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134 135 136 137

138 9. Sample panel composition: 139 A) Positive samples (Minimum n=80 for each sample type): These samples should be 140 clinical/spiked samples positive by reference real-time PCR assay and preferably represent all 141 genetic variants. The distribution of samples should be as follows: 142 Characteristic of positive sample Minimum no. of serum samples needed (for kits detecting CHPV in serum) Minimum no. of CSF samples needed (for kits detecting CHPV in CSF) A.1 Strong positive [Ct value ≤ 25] 24 24 A.2 Moderate positive [Ct value between >25 and ≤31] 28 28 A.3 Weak positive [Ct value

31 and ≤ 37] 28 28

143 For kits detecting CHPV in both serum and CSF, 80 positive serum samples and 80 positive CSF 144 samples should be used for performance evaluation. One sample type should not be substituted by 145 the other to reach the desired sample size in case there is paucity of samples. 146 Note: Since such large number of positive clinical samples may NOT be available for Chandipura virus, 147 pre-titrated and inactivated virus obtained from tissue culture fluid prepared in the laboratory will be used 148 to spike serum and CSF samples [dilution factor: 1:10 to 1:1000 to generate samples with different 149 intensities of positivity]. These spiked samples will be stored at -80°C, after being tested by the reference 150 assay.
151

152 B) Negative samples (n=80 for each sample type): All negative samples should be negative 153 by reference assay and CHPV IgM. Distribution of the negative samples should be as 154 follows: 155 Categories of samples as per the sample type Sample type Serum/plasma (Minimum n=80, (B.1 + B.2)) CSF (Minimum n=80, (B.1+B.2))

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B.1 Samples from cases of AES/ spiked samples which are RT-PCR positive for known pathogens but negative for CHPV (CHPV RNA and serology) 30

5 positive clinical/ spiked samples from each of the following diseases (confirmed by PCR):

Dengue virus @
Japanese Encephalitis @
HSV 1/2 *
West Nile Virus*
VSV *

Seven (07) positive clinical/ spiked samples from each of the following diseases:

a) Japanese Encephalitis @ b) Dengue virus @ c) HSV 1/2 * d) West Nile Virus *

Rabies virus (n=4)*
VSV (n=3)* B.2 Samples from cases with acute encephalitis and RT-PCR negative for the above-mentioned pathogens and CHPV (CHPV RNA and serology) 50 45 B.3 Healthy/ asymptomatic cases from endemic regions negative for CHPV (CHPV RNA and serology) 5 (desirable, not mandatory) 20 (desirable, not mandatory) Serum/plasma and CSF samples collected from the same case may be used for evaluation.

Cross reactivity panel is arranged in descending order of priority. The pathogens marked @ are essentially to be tested. It is recommended to test for all pathogens listed in the cross-reactivity panel. However, if there is an acute shortfall or non-availability of clinical samples, one may consider reducing only the pathogens of lower priority marked by * , while ensuring that the actual numbers of cross-reactive sample panel remain the same by compensating with the available “essentially to be tested” samples.

Testing for Rabies and VSV is recommended since both the viruses belong to the same family as Chandipura virus (Rhabdoviridae). Spiked specimens/ synthetic transcripts may be used for these viruses.

156 10. Evaluation method:
157 The index test and reference tests should be conducted simultaneously on the sample panel to 158 minimize the risk of false-negative results from the index test due to freeze-thaw cycles or sample 159 degradation from prolonged storage.
160 11. Interpretation of results:
161

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Reference test and index test results will be interpreted as per kit IFU.
162 12. Resolution of discrepant results: 163 True positive samples: These are samples positive by both the reference assay and index test. 164 True negative samples: These are samples negative by both the reference assay and index test. 165 False positive samples: These are samples negative by reference assay and positive by index test. 166 False negative samples: These are samples positive by reference assay and negative by index test. 167

168 13. Test reproducibility:
169 A) Sample size for lot-to-lot reproducibility: 170 Three lots of an assay shall be evaluated. Sample size for lot-to-lot reproducibility should be as 171 follows:
172 • First lot of the assay: should be tested on statistically significant number of positive and 173 negative samples as calculated in the protocol above
174 • Second lot of the assay: should be tested on 25 samples (15 positive samples comprising 175 10 low positives and 5 moderate/high positives, and 10 negative samples)
176 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 10 177 low positives and 5 moderate/high positives, and 10 negative samples)
178 If there is no lot-to-lot variation, accept the assay.
179 180

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181 14. Blinding of laboratory staff 182 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should be 183 blinded to the status of the clinical samples. The PI of the evaluation exercise should remain 184 unblinded, i.e., privy to the status of the samples. Another senior laboratory staff selected by the 185 PI may remain unblinded and carry out coding of samples and dispensing them into similar- 186 looking vials to be used for testing, and maintaining the database of results. Staff performing the 187 reference test and the test under evaluation, interpretation of the test result, and entering the results 188 against the coded samples in the database, should remain blinded to the status of samples till the 189 completion of evaluation. The data should be analyzed only by the PI of the evaluating lab. Refer 190 to Fig. 2. 191

192 193

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194

195 15. Acceptance criteria: 196 Expected sensitivity: ≥ 95%
197 Expected specificity: ≥ 98%
198 Cross-reactivity with other rhabdoviruses: Nil
199 Invalid test rate ≤5%
200

201 16. Publication Rights:
202 The PI(s) of the evaluating labs shall retain publication rights of the field evaluation as lead 203 author(s).
204

205 206 207 acceptable.
208 209 210 211 212

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213 214 215 216

217 VI. References
218

Sudeep AB, Gurav YK, Bondre VP. Changing clinical scenario in Chandipura virus 219 infection. Indian J Med Res. 2016;143(6):712-721. doi:10.4103/0971-5916.191929. 220
Sapkal GN, Sawant PM, Mourya DT. Chandipura Viral Encephalitis: A Brief Review. Open 221 Virol J. 2018 Aug 31;12:44-51. doi: 10.2174/1874357901812010044. PMID: 30288194; PMCID: 222 PMC6142667. 223
World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification- 224 Diagnostic assessment TGS-3.

Available at: 225 https://iris.who.int/bitstream/handle/10665/258985/WHO-EMP-RHT-PQT-TGS3-2017.03- 226 eng.pdf;sequence=1 227

228 VII. Performance Evaluation Report Format 229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

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PERFORMANCE EVALUATION REPORT FOR CHANDIPURA VIRUS REAL-TIME 244 PCR KITS 245 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: type,strong, moderate, weak) Negative samples (provide details, type,including cross reactivity panel)

246 Results 247

Reference assay ……….……………… (name)

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

Negative

Total

248

Estimate (%) 95% CI Sensitivity

Specificity

249 ● Conclusions: 250 o Cross reactivity with related viruses: 251 o Invalid test rate: 252 o Performance: Satisfactory / Not satisfactory 253

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(Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the 254 manufacturer from the batch mentioned above using ….. sample. Results should not be extrapolated to 255 other sample types.) 256

257 Disclaimers 258 259 260 Note: This report is exclusively for Chandipura………….. Kit (Lot No……) manufactured by …………… 261 (supplied by ……….) 262 263 264 Signature of Director/ Director-In-charge …………………… Seal ………………………………… 265 ****End of the Report 266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

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Annexure-1: Information on Operational and Test Performance Characteristics Required 285 from Manufacturers 286 The manufacturer should provide the following details about the IVD: 287

Instructions for Use 288
Scope of the IVD: to diagnose Chandipura virus 289
Intended Use Statement 290
Principle of the assay 291
Intended testing population (cases of Acute Febrile Illness/ AES) 292
Intended user (laboratory professional and/or health care worker at point-of-care) 293
Lot/batch No. 294
Date of manufacture 295
Date of Expiry 296
Information on operational Characteristics 297 i. Configuration of the kit/device 298 ii. Requirement of any additional equipment, device 299 iii. Requirement of any additional reagents 300 iv. Operation conditions 301 v. Storage and stability before and after opening 302 vi. Internal control provided or not 303 vii. Quality control and batch testing data 304 viii. Biosafety aspects- waste disposal requirements 305
Information on Test Performance Characteristics 306 i. Type of sample-CSF/Serum/Other specimen 307 ii. Volume of sample 308 iii. Any specific sample NOT to be tested 309 iv. Any additional sample processing required 310 v. Any additional device/consumable like sample transfer device, pipette, tube, etc required 311

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vi. Name of analyte to be detected 312 vii. Pathogen(s) targeted by the kit 313 viii. Time taken for testing 314 ix. Time for result reading and interpretation 315 x. Manual or automated (equipment) reading 316 xi. Limit of detection 317 xii. Diagnostic sensitivity 318 xiii. Diagnostic specificity 319 xiv. Stability and reproducibility 320 xv. Training required for testing 321 xvi. If yes, duration 322 xvii. Details of Cut-off and /or Equivocal Zone for interpretation of test 323 xviii. Interpretation of invalid and indeterminate results to be provided 324 xix. It is recommended to provide data demonstrating the precision 325 xx. Limit of detection 326

327 *Please mention “Not applicable” against sections not pertaining to the kit. 328

329 ****End of the Document 330

Page 0 of 17

1 STANDARD PERFORMANCE 2 EVALUATION PROTOCOL
3 DRAFT FOR STAKEHOLDER 4 COMMENTS 5

6 MULTIPLEX RESPIRATORY VIRUS 7 REAL TIME PCR 8 ICMR-CDSCO/IVD/GD/PROTOCOLS/09/2025 9

11 AUGUST, 2025 New Delhi, India

Multiplex Respiratory Virus Real Time PCR Kit Performance Evaluation Protocol ICMR-CDSCO/IVD/GD/PROTOCOLS/09/2025 Page 1 of 17

Table of Contents 12 S.N. Topic Page Number 1.
Performance evaluation protocol for Multiplex Respiratory Virus real-time PCR kits 2 2.
Performance Evaluation Report Format 13 3.
Information on Operational and Test Performance Characteristics Required from Manufacturers 15

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Performance evaluation protocol for multiplex respiratory virus real-time PCR kit 38 I. Background:
39 CDSCO and ICMR, New Delhi, have aimed at facilitating the availability of Quality-Assured 40 diagnostic kits appropriate for use in India. Hence the following guidelines shall establish the 41 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 42 evaluation is to independently verify the manufacturer’s claim regarding IVD performance. 43 This recommendation focuses on the laboratory performance evaluation of multiplex respiratory 44 virus real time PCR kit. All clinical samples tested in the study should be evaluated in accordance 45 with the candidate test’s instructions for use.
46 II. Purpose: 47 To evaluate the performance characteristics of multiplex respiratory virus real-time PCR kits using 48 irreversibly de-identified leftover archived clinical/spiked samples. 49 III. Scope of the document: 50 This document outlines performance evaluation protocol for multiplex real time PCR assays 51 detecting the following respiratory viruses of utmost importance in human clinical specimens 52 (Table 1), as determined by ICMR appointed working group and expert group of physicians and 53 clinical microbiologists following extensive literature review and real-life experience. This 54 pathogen list has been developed as part of the National One Health Mission. 55 Table 1: List of respiratory viruses within the scope of this performance evaluation protocol 56

```
Influenza virus A 
```
Influenza virus B
SARS Coronavirus-2
```
Respiratory syncytial virus 
```
```
 Adenovirus 
```

Human Respiroviruses 1 and 3 and Human Rubulaviruses 2 and 4 (erstwhile Human

Parainfluenzaviruses 1-4) 7. Human metapneumovirus
8. Measles virus 9. Rhinovirus 10. Human Bocavirus 11. Enterovirus 12. Cytomegalovirus

57 IV. Requirements:
58

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Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 59 the kit to be evaluated works in a closed system format, the manufacturer needs to supply 60 the required equipment. 61
Evaluation sites/laboratories (With required equipment) 62
Reference test kits 63
Characterised Evaluation panel 64
Laboratory supplies
65 V. Ethical approvals:
66 Performance evaluation activities using irreversibly de-identified leftover clinical samples are 67 exempt from ethics approval as per ICMR’s Guidance on Ethical Requirements for 68 Laboratory Validation Testing, 2024.
69 Investigators are required to submit a self-declaration form, as outlined in the ICMR guidelines, 70 to the institutional authorities and ethics committee for information. 71 VI. Procedure: 72
Study design/type: Diagnostic accuracy study using irreversibly de-identified archived/ 73 spiked clinical samples
74 75 Identified IVD kit evaluation laboratories should be well-equipped and establish their 76 proficiency through ALL of the following: 77 A. Accreditation at least one of the Quality management systems for at least one respiratory viral 78 pathogen molecular testing (accreditation for Testing Lab / Calibration Lab as per ISO 17025, 79 Medical Lab as per ISO 15189, PT provider as per ISO/IEC 17043), or CDSCO approved 80 Reference laboratory.
81 B. Staff training: All the staff involved in IVD evaluation should undergo hands-on training and 82 competency testing on the following:
83 ⮚ Preparation & characterization of reference sample panel 84 ⮚ Handling of multiplex respiratory virus RT-PCR kits received for performance evaluation 85 (Verification/Storage/Unpacking etc). 86 ⮚ Testing 87 ⮚ Data handling, data safety & confidentiality 88
Preparation of multiplex respiratory virus evaluation panel 89 A well characterised panel of positive and negative clinical samples is a critical requirement for 90 evaluation of these RT-PCR IVD kits. Also, a statistically significant number of clinical samples 91 should be used for the evaluation. 92

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The sample type for respiratory virus detection is usually nasopharyngeal/oropharyngeal swab. If 93 a kit claims to detect these viruses across several sample types, attempt should be made to evaluate 94 the assay across all the sample types. In case all the sample types mentioned in the IFU are not 95 available with the lab, the performance evaluation report should clearly mention the sample type 96 against which the kit is evaluated. There should be no ambiguity about the type of sample used for 97 evaluation. 98 4. Nucleic acid extraction 99 Nucleic acid extraction should be performed using standard techniques. If the manufacturer of the 100 index test recommends a specific nucleic acid extraction kit, it needs to be provided by the 101 manufacturer if the evaluation lab is unable to procure the same.
102 *Caution is advised in the selection of a nucleic acid extraction kit since the target pathogens comprise 103 both RNA and DNA viruses.
104 5. Real-Time PCR System 105 PCR should be performed using IVD-approved machines. If any equipment(s) is specified in the 106 IFU of the index test, it should be used for the evaluation, and it should be provided by the 107 manufacturer if not available within the lab’s IVD evaluation scope.
108 Real-time closed systems/devices awaiting evaluation should be provided by the manufacturer 109 along with all necessary components, supplies and reagents. 110 6. Internal control/Extraction control 111 The test under evaluation (index test) must have an internal control (housekeeping gene), with or 112 without an extraction control (nucleic acid added before extraction to a sample). 113 7. Reference assay:
114 The following points are to be noted: 115 i.A WHO Pre-Qualified/ US FDA/ ATAGI Australia/ PMDA Japan approved single plex (for a 116 particular target pathogen) or multiplex real-time PCR assay/ ICMR-NIV Pune in-house single 117 plex (for a particular target pathogen) or multiplex Real Time PCR Assay should be used as the 118 reference assay. 119 ii.Since the list of target pathogens is extensive, a combination of single plex and/or multiplex 120 assays may be used as the reference assay(s), as long as these reference assays satisfy the criteria 121 outlined in point 7(i).
122 All samples positive for a particular pathogen should be confirmed positive by the reference assay. 123 All samples negative for a particular pathogen should be confirmed negative by the reference 124 assay. 125

126

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Sample size for performance evaluation: The 2009 FDA guidance document 127 “Respiratory Viral Panel Multiplex Nucleic Acid Assay - Class II Special Controls 128 Guidance for Industry and FDA Staff”, recommends including a sufficient number of 129 prospectively collected samples for each specimen type to generate a result with at least 130 90% sensitivity with a lower bound of the two-sided 95% confidence interval (CI) greater 131 than 80, and demonstrate specificity with a lower bound of the two-sided 95% CI greater 132 than 90%. In accordance with these guidelines and for feasibility of evaluation of these 133 extensive multiplex panels, sample size for each pathogen is calculated assuming ≥90% 134 sensitivity and specificity of the index test, 95% confidence level, absolute precision of 135 7.5%, and ≤5% invalid test rate. A minimum of 65 positive clinical samples (rounded to 136

and a minimum of 65 negative clinical samples for each target pathogen are required 137 for performance evaluation of the assay. However, 120 negative samples are recommended 138 per pathogen to account for an extensive cross reactivity panel. Sample sizes are calculated 139 using the formulae: 140

141 𝑑2 x (1 −IR)
142 𝑑2 x (1 −IR)
143

144

145 146 147 148 149 150 151 152 153 The details of sample requirement are outlined in Table 2.
154

155 Table 2: No. of samples required for performance evaluation: 156 Pathogen Minimum no. of positive samples needed per pathogen Minimum no. of negative samples recommended per pathogen

Influenza virus A* 70 120
Influenza virus B* 70 120
SARS Coronavirus-2 70 120

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Respiratory syncytial virus* 70 120
Adenovirus* 70 120
Human Respirovirus 1 and Human Respirovirus 3 and Human Rubulavirus 2 and Human Rubulavirus 4* 70 120
Human metapneumovirus * 70 120
Measles virus 70 120
Rhinovirus** 70 120
Human Bocavirus 70 120
Enterovirus** 70 120
Cytomegalovirus 70 120 *If a kit claims to differentiate between virus types/subtypes, please use minimum 70 positive samples and minimum 120 negative samples for each virus type/subtype. If such type/subtype specific samples are not available (only for predicate device) or if the kit does not claim to differentiate between pathogen types/subtypes, and the kit is evaluated against the pathogen as a whole, the reports should be issued with a disclaimer that performance characteristics against pathogen types/subtypes have not been evaluated separately. However, in such a scenario, the evaluating centre should try to include all types/subtypes of the pathogen in the evaluation panel (even if the numbers are not statistically significant for each pathogen type).

**If clinical samples positive separately for Rhinovirus/Enterovirus are not available (only for predicate device), or if the kit does not differentiate between Enteroviruses and Rhinoviruses, please use minimum 70 samples positive for Rhinovirus/Enterovirus in the positive sample panel and issue the reports with a disclaimer that performance characteristics against Rhinovirus/Enterovirus have not been evaluated separately.

Influenza virus, SARS Coronavirus 2, Respiratory Syncytial Virus and Human Metapneumovirus positive samples used for evaluation should have been collected within the past 1 year.

157 Notes for Table 2: 158

Samples positive for currently circulating virus strains should be used in the positive 159 sample panel, with representation from all virus types/subtypes. 160
Sample positive for a particular virus type and negative for the target pathogen being 161 considered may be used in the negative sample panel for the target pathogen, e.g.: a sample 162 positive for SARS-CoV-2 may be used as a negative sample for RSV. 163

164 9. Sample panel composition: 165 A. Human samples 166 A.1 Positive samples for each pathogen/ type or subtype of pathogen (Minimum 167 n=70): Clinical samples positive by the reference real-time PCR assay should be included, 168 as per the following criteria
169

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A.1.1 Strong positive (Ct value <25) = 20 samples
170 A.1.2. Moderate positive (Ct value between 25-30) = 25 samples 171 A.1.3 Weak positive (Ct value >30-36) = 25 samples
172 A.2 Negative samples for each pathogen/ type or subtype of pathogen (Minimum 173 n=120): All negative samples should be negative for the target pathogen/ its type or 174 subtype by the reference real-time PCR assay. Distribution of the negative samples should 175 be as follows: 176 A.2.1 NP/OP swab from individuals with respiratory infection that are negative for the 177 target pathogen/its type or subtype = 35 samples ** 178 A.2.2 NP/OP swab from apparently healthy individuals with no respiratory symptoms = 179 23 samples ** 180 A.2.3 Cross reactivity panel (Table 3): Samples negative for the target pathogen but 181 positive for other common respiratory viruses = 62 samples *** 182 Archived frozen sample aliquots if used for the evaluation, should not be thawed more than 183 once.
184 ** If samples are available with the evaluating lab that satisfy these criteria and are negative for 185 all the pathogens targeted by the kit, the same samples may be included in the negative sample 186 panel for all target pathogens to prevent wastage of resources.
187 *** Same positive samples may be included in the cross-reactivity panel of several target 188 pathogens to prevent wastage of resources e.g.: the same Influenza A virus positive sample may 189 be included in the cross-reactivity panel for RSV, Human Metapneumovirus, SARS-CoV-2 etc. 190

191 Table 3: Cross reactivity panel for performance evaluation of multiplex respiratory virus 192 real time PCR kit 193

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Target Pathogen Virus-wise no. of samples needed for cross reactivity analysis Total no. of cross reacti ve sampl es per patho gen Influe nza virus A * Influe nza virus B* SARS Coronav irus-2 * Respira tory syncyti al virus * Adenov irus @ Human Respirovi ruses 1 and 3 , Human Rubulavir uses 2 and 4 # Human metapneumov irus@ Meas les virus * Rhinovir us@ $ Huma n Bocav irus Enterov irus $ Cytomegalo virusᴥ Seasonal coronavir uses* Rube lla

Influenza virus A 0 5 5 5 5 5 5 5 5 5 5 5 5 2 62
Influenza virus B 5 0 5 5 5 5 5 5 5 5 5 5 5 2 62
SARS Coronavirus-2 5 5 0 5 5 5 5 5 5 5 5 5 5 2 62
```
Respiratory 
```

syncytial virus 5 5 5 0 5 5 5 5 5 5 5 5 5 2 62 5. Adenovirus 5 5 5 5 0 5 5 5 5 5 5 5 5 2 62 6. Human Respi roviruses 1 and 3, Human Rubulaviruses 2 and 4
5 5 5 5 5 0 5 5 5 5 5 5 5 2 62 7. Human metapneumovirus
5 5 5 5 5 5 0 5 5 5 5 5 5 2 62 8. Measles virus 5 5 5 5 5 5 5 0 5 5 5 5 5 2 62 9. Rhinovirus 5 5 5 5 5 5 5 5 0 5 5 5 5 2 62 10. Human Bocavirus 5 5 5 5 5 5 5 5 5 0 5 5 5 2 62 11. Enterovirus 5 5 5 5 5 5 5 5 5 5 0 5 5 2 62 12. Cytomegalovirus 5 5 5 5 5 5 5 5 5 5 5 0 5 2 62 *Include all currently circulating strains/types/subtypes @It is desirable to have representation from all types of the pathogen, since even approved assays may not always differentiate between pathogen types.

Include at least 1 of each

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$ If clinical samples positive separately for Rhinovirus/Enterovirus are not available, please use total 10 samples positive for Rhinovirus/Enterovirus in the cross-reactivity panel for remaining pathogens. ᴥ Can use lower respiratory tract specimen

If a kit claims to differentiate between virus types/subtypes, please use 5 positive samples for each virus type in the cross reactivity panel for other target pathogens. If such type specific samples are not available and the kit is evaluated against the pathogen as a whole, it should be clearly mentioned in the report.

If available, samples positive for relevant bacterial pathogens and other relevant viruses (with which majority of the population is likely to be infected), should also be included in the cross-reactivity panel. Influenza virus, SARS Coronavirus 2, Respiratory Syncytial Virus and Human Metapneumovirus positive samples used for evaluation should have been collected within the past 1 year.

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B. Contrived samples: 194 Contrived positive and negative samples may be used for evaluation in case of 195 paucity/unavailability of human clinical samples. Positive contrived samples should be 196 positive and negative contrived samples should be negative for the target 197 pathogen/type/subtype using the reference assay. The number and distribution of positive 198 and negative samples, including the cross reactivity panel, should remain the same. 199 Contrived positive samples (as part of positive sample panel/ cross-reactivity panel) should 200 be prepared by spiking a sample matrix negative for the pathogen with a pathogen-infected 201 cell line, genomic DNA plasmids or RNA transcripts. 202 It is recommended to demonstrate equivalence between contrived and clinical specimens. 203 Serial dilutions of clinical sample and serial dilutions of contrived sample with targeted 204 levels of analyte should be compared for demonstrating equivalence. 205 10. Evaluation method: 206 The index test and the reference assay should be run simultaneously on the sample panel, 207 and results should be recorded.
208

209 11. Interpretation of results:
210 Reference test and index test results will be interpreted as per kit IFU.
211 12. Resolution of discrepant results: 212 True positive samples: These are samples positive by reference assay and index test. 213 True negative samples: These are samples negative by reference assay and index test. 214 215 index test. 216 217 index test. 218 13. Test reproducibility 219 A. Sample size for lot-to-lot reproducibility 220 Three lots of an assay should be evaluated. Sample size for lot-to-lot reproducibility should be 221 as follows:
222 223 and negative samples for each pathogen/type of pathogen as calculated in the protocol.
224 • Second lot of the assay: should be tested on 25 samples for each pathogen/type of 225 pathogen (15 positive samples comprising 10 low positive AND 5 moderate/high 226 positive samples, and 10 negative samples).
227 • Third lot of the assay: should be tested on 25 samples for each pathogen/type of 228 pathogen (15 positive samples comprising 10 low positive AND 5 moderate/high 229 positive samples, and 10 negative samples). 230 • There should be no lot-to-lot variability. 231

232

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Blinding of laboratory staff 233 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should 234 be blinded to the status of the clinical samples. The PI of the evaluation exercise should 235 remain unblinded, i.e., privy to the status of the samples. Another senior laboratory staff 236 selected by the PI may remain unblinded and carry out coding of samples and dispensing 237 them into similar-looking vials to be used for testing, and maintaining the database of 238 results. Staff performing the reference test and the test under evaluation, interpretation of 239 the test result, and entering the results against the coded samples in the database, should 240 remain blinded to the status of samples till the completion of evaluation. The data should 241 be analyzed only by the PI of the evaluating lab. Refer to Fig. 2. 242

243 244

245

246

247 15. Acceptance Criteria 248 Expected sensitivity for each pathogen/type/subtype: ≥90% 249 Expected specificity for each pathogen/type/subtype: ≥95% 250 Cross reactivity with other viruses as outlined in the negative sample panel: Nil 251 252

253

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 16. Publication Rights:

254 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead author(s). 255

256 257 258 acceptable.
259 260 261 262 263 264 265 266 267

268 VII. References: 269 1. Food and Drug Administration. Respiratory Viral Panel Multiplex Nucleic Acid Assay - Class 270 II Special Controls Guidance for Industry and FDA Staff. Available at: 271 https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation- 272 emitting-products/respiratory-viral-panel-multiplex-nucleic-acid-assay-class-ii-special- 273 controls-guidance-industry-and [Accessed on 22nd January, 2025]. 274 2. Food and Drug Administration. 510(k) Substantial Equivalence Determination Decision 275 Summary, Biofire Diagnostics LLC, FilmArray Pneumonia Panel. Available at: 276 https://www.accessdata.fda.gov/cdrh_docs/reviews/K180966.pdf [Accessed on 19th January 277 2025] 278 3. Food and Drug Administration: Testing for Human Metapneumovirus (hMPV) Using Nucleic 279 Acid Assays - Class II Special Controls Guidance for Industry and FDA Staff. 2009. Available 280 at: https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation- 281 emitting-products/testing-human-metapneumovirus-hmpv-using-nucleic-acid-assays-class-ii- 282 special-controls-guidance#3 [Accessed on January 11, 2025] 283

284 VIII. Performance evaluation report format 285

286

287

288

289

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PERFORMANCE EVALUATION REPORT FOR MULTIPLEX RESPIRATORY VIRUS 290 REAL-TIME PCR KITS 291 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Pathogens detected by the assay Intended Use

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

292 Results: Tables 1 and 2 should be made for each pathogen/type of pathogen targeted by the kit 293 under evaluation 294

295 Table 1: 2x2 table for sensitivity and specificity calculation (prepare 1 table for each target pathogen /type/ 296 subtype) 297

298

Reference assay ……….……………… (name)

Positive Negative Total Name of …….. virus real-time PCR Positive

Negative

Total

299

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Table 2: Sensitivity and specificity 300

301

Estimate (%) 95% CI Sensitivity

Specificity

302 ● Details of cross reactivity with other viruses in the cross-reactivity panel: 303 ● Conclusions: 304 o Sensitivity, specificity 305 o Cross reactivity 306 o Invalid test rate 307 o Performance: Satisfactory / Not satisfactory 308 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 309 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 310 Disclaimers 311 312 313 Note:
314 This report is exclusively for Human Metapneumovirus………….. Kit (Lot No……) manufactured by 315 …………… (supplied by ……….) 316 317 318 Signature of Director/ Director-In-charge …………………… Seal …………………………. 319 ****End of the Report 320

321

322

323

324

325

326

327

328

329

330

331

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Annexure-1: Information on Operational and Test Performance Characteristics Required 332 from Manufacturers 333 1. The manufacturer should provide the following details about the IVD: 334 2. Instructions for Use 335 3. Scope of the IVD:
336 4. Pathogens/type/subtype of pathogens targeted by the kit 337 5. Intended Use Statement 338 6. Principle of the assay 339 7. Intended testing population (cases of ARI/ILI/SARI) 340 8. Intended user (laboratory professional and/or health care worker at point-of-care) 341 9. Lot/batch No. 342 10. Date of manufacture 343 11. Date of Expiry 344 12. Information on operational Characteristics 345 i. Configuration of the kit/device 346 ii. Requirement of any additional equipment, device 347 iii. Requirement of any additional reagents 348 iv. Operation conditions 349 v. Storage and stability before and after opening 350 vi. Internal control provided or not 351 vii. Quality control and batch testing data 352 viii. Biosafety aspects- waste disposal requirements 353 11. Information on Test Performance Characteristics 354 i. Type of sample-NP/OP swab, other respiratory specimen 355 ii. Volume of sample 356 iii. Any specific sample NOT to be tested 357 iv. Any additional sample processing required 358 v. Any additional device/consumable like sample transfer device, pipette, tube, etc required 359 vi. Name of analyte to be detected 360

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vii. Pathogens targeted by the kit 361 viii. Time taken for testing 362 ix. Time for result reading and interpretation 363 x. Manual or automated(equipment)reading 364 xi. Limit of detection 365 xii. Diagnostic sensitivity 366 xiii. Diagnostic specificity 367 xiv. Stability and reproducibility 368 xv. Training required for testing 369 xvi. If yes, duration 370 xvii. Details of Cut-off and /or Equivocal Zone for interpretation of test 371 xviii. Interpretation of invalid and indeterminate results to be provided 372 xix. It is recommended to provide data demonstrating the precision 373 xx. Limit of detection 374 *Please mention “Not applicable” against sections not pertaining to the kit. 375

376

377 ***End of the Document 378

1 DENGUE IgG BASED ASSAYS 2 ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 3

6 STANDARD PERFORMANCE EVALUATION PROTOCOLS
DRAFT FOR STAKEHOLDER COMMENTS

AUGUST, 2025 New Delhi, India

Dengue IgG Based Assays Performance Evaluation Protocols ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 1 | Page

Table of Contents 7 S.No. Content Page Number 1.
Performance evaluation protocol for Dengue IgG RDT kits 2 2.
Performance evaluation protocol for Dengue IgM and IgG RDT combo kits 13 3.
Performance evaluation protocol for Dengue IgG ELISA kits 26 4.
Information on Operational and Test Performance Characteristics Required from Manufacturers
37

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Performance evaluation protocol for Dengue IgG RDT kits 31 I. Background:
32 CDSCO/ICMR, New Delhi, have aimed at facilitating the availability of Quality-Assured 33 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish the 34 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 35 evaluation is to independently verify the manufacturer’s claim regarding in-vitro diagnostic kit 36 (IVD) performance. 37 II. Purpose: 38 To evaluate the performance characteristics of Dengue IgG RDT kits in the diagnosis of primary 39 and secondary dengue infections using irreversibly de-identified leftover archived clinical 40 samples. 41 III. Requirements:
42 a) Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 43 the kit to be evaluated works in a closed system format, the manufacturer needs to supply 44 the required equipment. 45 b) Evaluation sites/laboratories (With required equipment) 46 c) Reference test kits 47 d) Characterised Evaluation panel 48 e) Laboratory supplies
49 IV. Ethical approvals:
50 Performance evaluation activities using irreversibly de-identified leftover clinical samples are 51 exempt from ethics approval as per ICMR’s Guidance on Ethical Requirements for Laboratory 52 Validation Testing, 2024.
53 Investigators are required to submit a self-declaration form, as outlined in the ICMR guidelines, 54 to the institutional authorities and ethics committee for information. 55 V. Procedure: 56

Study design/type: Diagnostic accuracy study using irreversibly de-identified archived/ 57 spiked leftover clinical samples
58 59 Identified IVD kit evaluation laboratories should establish their proficiency through 60 ALL of the following: 61 A. Accreditation for at least one of the Quality management systems (accreditation for Testing 62 Lab / Calibration Lab (ISO: 17025), Medical Lab (ISO: 15189), PT provider (ISO: 17043) or 63 CDSCO approved Reference laboratory. 64

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B. Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 65 and competency testing on following
66 ⮚ Preparation & characterization of kit evaluation panel
67 ⮚ Handling of Dengue IgG Rapid IVD kits received for performance evaluation 68 (Verification/Storage/Unpacking etc). 69 ⮚ Testing, interpreting, recording of results & reporting 70 ⮚ Data handling, data safety & confidentiality 71

72 3. Preparation of Dengue IgG Rapid IVD kit evaluation panel 73 Well characterised Dengue IVD kit evaluation panel is a critical requirement for performance 74 evaluation of IVD kits. Hence statistically significant number of sera samples should be 75 collected from Dengue NS1/PCR/IgM confirmed cases. Further characterised for Dengue IgG 76 positivity by using approved reference kits having high sensitivity and specificity. 77 Dengue IgG performance evaluation panel need to be tested again by the reference assays at 78 the time of evaluating a particular index test to confirm the positive and negative status of the 79 samples. 80 4. Reference assay:
81 Positive and negative samples should be characterized using composite reference standard 82 of Dengue IgG AND one additional marker of Dengue (NS1 or IgM or PCR). The 83 following kits should be used for characterization of the sample panel: 84 • Panbio Dengue IgG capture ELISA kit 85 • WHO Pre-Qualified/ US-FDA/ ATAGI Australia/ PMDA Japan approved Dengue IgM 86 ELISA kit 87 • NS1 antigen status to be assessed using WHO Pre-Qualified/ US-FDA/ ATAGI Australia/ 88 PMDA Japan approved NS1 ELISA kit 89 • Serotype status to be assessed using a combination of CDC/NIV real-time PCR serotyping 90 protocols.
91 5. Sample size for performance evaluation: 92

93 Sample sizes of positive and negative samples and sample panel composition against different 94 values of sensitivity and specificity are provided in Tables 1 and 2. Sample sizes have been 95 calculated assuming 95% level of significance, an absolute precision of 5%, and invalid test 96 rate ≤5% using the following formulae: 97

98 𝑑2 x (1 −IR)
99

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 4 | Page

𝑑2 x (1 −IR)
100

101

102 103 104 105 106 107 108 109 110

111

112 Appropriate sample size has to be chosen from the tables according to the values of sensitivity and 113 specificity being claimed by the manufacturer. If a claimed sensitivity/specificity is not present in 114 the table, the manufacturer needs to consider the sample size associated with the largest 115 sensitivity/specificity provided in the table that is smaller to the claimed value (that is, as per the 116 next smaller value of the sensitivity/ specificity available in the table). For example, if a 117 manufacturer claims a sensitivity of 93%, they are required to use a sample size mentioned against 118 90% sensitivity. Similarly, a claim of 87% specificity would require usage of the sample size 119 outlined for 85% specificity.
120 Positive samples: The panel of positive samples should include samples positive for IgG by the 121 reference assay. The samples should also be positive for either dengue NS1 antigen or dengue IgM 122 antibodies.
123 Negative samples: Samples which are negative by reference dengue IgG test should form the 124 negative sample panel.
125 Table 1. Sample sizes and panel composition of positive dengue IgG samples for different values 126 of sensitivity claimed by the manufacturer. 127 Sensitivity Calculated sample size Minimum no. of Positive Samples required [Sample size rounded off] # Sample Panel Composition 99% 16 20 Strong Positive: 6 Moderate Positive: 7 Weak Positive: 7

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95% 77 80 Strong Positive: 24 Moderate Positive: 28 Weak Positive: 28 90% 145 150 Strong Positive: 44 Moderate Positive: 53 Weak Positive: 53 85% 206 210 Strong Positive: 62 Moderate Positive: 74 Weak Positive: 74 80% 258 260 Strong Positive: 78 Moderate Positive: 91 Weak Positive: 91 The samples need to be classified as strong, moderate and weak positives based on ELISA units of the reference assay.

128

129 Table 2. Sample sizes and panel composition of negative dengue IgG samples for different values 130 of specificity claimed by the manufacturer. 131 Specificity Calculated sample size Minimum no. of Negative Samples required [Sample size rounded off]

Sample Panel Composition 99%# 16 20 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 7
2.Acute febrile illness cases: 8
• Chikungunya positive samples:2 • Dengue (NS1 & IgM & IgG & PCR) negative samples:6 3.Samples from other flavivirus disease cases (cross- reactive panel): 3 • Japanese Encephalitis IgM/IgG positive: 1 @ • West Nile Virus IgM/IgG positive: 1 * • Zika Virus IgM/IgG positive: 1 * 4. aHealthy subjects from endemic regions: 2
95% 77 80 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 27
2.Acute febrile illness cases: 32

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• Chikungunya positive samples:8 negative samples:24 3.Samples from other flavivirus disease cases(cross- reactive panel): 9 • Japanese Encephalitis IgM/IgG positive: 3 @ • West Nile Virus IgM/IgG positive: 3 * • Zika Virus IgM/IgG positive: 3 * 4. aHealthy subjects from endemic regions: 12
90% 145 150 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 50
2.Acute febrile illness cases: 60
• Chikungunya positive samples:15 negative samples:45 3.Samples from other flavivirus disease cases(cross- reactive panel): 15 • Japanese Encephalitis IgM/IgG positive: 5 @ • West Nile Virus IgM/IgG positive: 5 * • Zika Virus IgM/IgG positive: 5 * 4. aHealthy subjects from endemic regions: 25
85% 206 210 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 70
2.Acute febrile illness cases: 84
• Chikungunya positive samples:21 negative samples:63 3.Samples from other flavivirus disease cases(cross- reactive panel): 21 • Japanese Encephalitis IgM/IgG positive: 7 @ • West Nile Virus IgM/IgG positive: 7 * • Zika Virus IgM/IgG positive: 7 * 4. aHealthy subjects from endemic regions: 35
80% 258 260 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 85
2.Acute febrile illness cases: 104
• Chikungunya positive samples:26 negative samples:78 3.Samples from other flavivirus disease cases(cross- reactive panel): 27 • Japanese Encephalitis IgM/IgG positive: 9 @ • West Nile Virus IgM/IgG positive: 9 * • Zika Virus IgM/IgG positive: 9 * 4. aHealthy subjects from endemic regions: 44

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 7 | Page

a Samples from healthy subjects from endemic regions negative for all dengue markers (NS1, IgM, IgG, RNA)

Cross reactivity panel is arranged in descending order of priority. The pathogens marked @ are essentially to be tested. It is recommended to test for all pathogens listed in the cross-reactivity panel. However, if there is an acute shortfall or non-availability of clinical samples, one may consider reducing only the pathogens of lower priority marked by * , while ensuring that the actual numbers of cross-reactive sample panel remain the same by compensating with the available “essentially to be tested” samples.

Note: If IgM/IgG positive samples for cross reactive flaviviruses are not available, commercially available IgM/IgG sera panel for different viruses can be procured and used to test cross reactivity.

132

133 6. Evaluation method: 134 The index test and the reference assay should be run simultaneously on the sample panel, 135 and results should be recorded.
136

137

138 7. Interpretation of results:
139 Reference test and index test results will be interpreted as per kit IFU.
140 8. Resolution of discrepant results: 141 True positive samples: These are samples positive by reference assay and index test. 142 True negative samples: These are samples negative by reference assay and index test. 143 144 index test. 145 146 index test. 147

148 9. Test reproducibility 149 A. Sample size for lot-to-lot reproducibility 150 Three lots of an assay shall be evaluated. Sample size for lot-to-lot reproducibility should be 151 as follows:
152 153 and negative samples as calculated in the protocol.
154

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• Second lot of the assay: should be tested on 25 samples (15 positive samples 155 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 156 samples).
157 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 158 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 159

160 161

162

163

164 B. Sample size for reader-to-reader reproducibility 165 For reader-to-reader reproducibility, sample size should be 25 (15 positive samples comprising 10 166 low positive AND 5 moderate/high positive samples, and 10 negative samples). 167

168 Two operators will be reading the test results independently as per manufacturer’s instruction. 169 Agreement should be 100% between the operators. 170 10. Blinding of laboratory staff 171 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should 172 be blinded to the status of the clinical samples. The PI of the evaluation exercise should 173 remain unblinded, i.e., privy to the status of the samples. Another senior laboratory staff 174 selected by the PI may remain unblinded and carry out coding of samples and dispensing 175 them into similar-looking vials to be used for testing, and maintaining the database of 176

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results. Staff performing the reference test and the test under evaluation, interpretation of 177 the test result, and entering the results against the coded samples in the database, should 178 remain blinded to the status of samples till the completion of evaluation. The data should 179 be analyzed only by the PI of the evaluating lab. Refer to Fig. 2. 180

181 182

183

184

185 11. Acceptance Criteria
186 Expected sensitivity: ≥80% 187 Expected specificity: ≥90% 188 Cross reactivity: Nil 189 190 To achieve at least the performance characteristics outlined in the acceptance criteria, ≥260 191 positive samples and ≥150 negative samples should be used for evaluation. 192 12. Publication Rights: 193 The PI (s) of the evaluating labs shall retain publication rights of the evaluation as lead author(s). 194

195

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196 197 acceptable.
198 199 200 201 202 203 204 205 206

207 VI. References: 208

Vazquez S, Hafner G, Ruiz D, Calzada N, Guzman MG. Evaluation of immunoglobulin M and G capture 209 enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue infections. J Clin Virol. 2007 210 Jul;39(3):194-8. doi: 10.1016/j.jcv.2007.04.003..
211
WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. (Diagnostics 212 evaluation series, 3). ISBN 978 92 4 159775 3.
213
Central Drugs Standard Control Organization. Guidance on Performance Evaluation of In-vitro Diagnostic 214 Medical Devices. 2018. Available at: https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf- 215 documents/medical device/guidanceperformanceivd.pdf 216
Central Drugs Standard Control Organization. In-Vitro Diagnostic (IVD) Medical Devices Frequently 217 Asked Questions. 2022. Available at: https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf- 218 documents/IVD/FAQs/CDSCO-IVD-FAQ-03-2022-.pdf
219
U.S. Food and Drug Administration. Dengue Virus Serological Reagents - Class II Special Controls 220 Guideline for Industry and Food and Drug Administration Staff.

Available at: 221 https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation-emitting- 222 products/Dengue-virus-serological-reagents-class-ii-special-controls-guideline-industry-and-food-and- 223 drug
224 6. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – Diagnostic 225 Assessment TGS-3. 2017. Available at: https://iris.who.int/bitstream/handle/10665/258985/WHO-EMP- 226 RHT-PQT-TGS3-2017.03-eng.pdf;sequence=1
227

228 *The validation protocols need to be revisited after introduction of Dengue vaccines and the 229 acceptance criteria needs revisiting every year so as to enable the availability of best 230 diagnostic kits.
231

232 VII. Performance evaluation report format 233

234

235

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PERFORMANCE EVALUATION REPORT FOR DENGUE IgG RDT KIT 236 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

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

237 Results:
238

Reference assay ……….……………… (name)

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

Negative

Total

239

Estimate (%) 95% CI Sensitivity

Specificity

Conclusions: 240 o Sensitivity, specificity 241 o Cross-reactivity: 242 o Invalid test rate: 243

244 o Performance: Satisfactory / Not satisfactory 245

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(Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 246 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 247 Disclaimers 248 249 250 Note: This report is exclusively for ………………………Kit (Lot No……) manufactured by …………… 251 (Supplied by ……….) 252 253 254 Signature of Director/ Director-In-charge …………………… Seal ………………………. 255

256 ****End of the Report 257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

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Performance evaluation protocol for Dengue IgM and IgG RDT combo kits 276 I. Background:
277 CDSCO/ICMR, New Delhi, have aimed at facilitating the availability of Quality-Assured 278 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish the 279 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 280 evaluation is to independently verify the manufacturer’s claim regarding in-vitro diagnostic kit 281 (IVD) performance. 282 II. Purpose: 283 To evaluate the performance characteristics of Dengue IgM and IgG RDT combo kits in the 284 diagnosis of dengue and discriminating primary and secondary dengue infections using 285 irreversibly de-identified leftover archived clinical samples. 286 III. Requirements:
287 f) Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 288 the kit to be evaluated works in a closed system format, the manufacturer needs to supply 289 the required equipment. 290 g) Evaluation sites/laboratories (With required equipment) 291 h) Reference test kits 292 i) Characterised Evaluation panel 293 j) Laboratory supplies
294 IV. Ethical approvals:
295 Performance evaluation activities using irreversibly de-identified leftover clinical samples are 296 exempt from ethics approval as per ICMR’s Guidance on Ethical Requirements for Laboratory 297 Validation Testing, 2024.
298 Investigators are required to submit a self-declaration form, as outlined in the ICMR guidelines, 299 to the institutional authorities and ethics committee for information. 300 V. Procedure: 301

Study design/type: Diagnostic accuracy study using irreversibly de-identified archived/ 302 spiked leftover clinical samples 303 304 Identified IVD kit evaluation laboratories should establish their proficiency through 305 ALL of the following: 306 A.Accreditation for at least one of the Quality management systems (accreditation for Testing 307 Lab / Calibration Lab (ISO: 17025), Medical Lab (ISO: 15189), PT provider (ISO: 17043) or 308 CDSCO approved Reference laboratory. 309

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B.Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 310 and competency testing on following
311 ⮚ Preparation & characterization of kit evaluation panel
312 ⮚ Handling of Dengue IgM and IgG Rapid IVD kits received for performance evaluation 313 (Verification/Storage/Unpacking etc). 314 ⮚ Testing, interpreting, recording of results & reporting 315 ⮚ Data handling, data safety & confidentiality 316

317 3. Preparation of Dengue IgM and IgG Rapid IVD kit evaluation panel 318 Well characterised Dengue IVD kit evaluation panel is a critical requirement for performance 319 evaluation of IVD kits. Hence statistically significant number of sera samples should be 320 collected from Dengue NS1/PCR/IgM confirmed cases. Further characterised for Dengue IgG 321 positivity by using approved reference kits having high sensitivity and specificity. 322 Dengue IgG performance evaluation panel need to be tested again by the reference assays at 323 the time of evaluating a particular index test to confirm the positive and negative status of the 324 samples. 325 4. Reference assay:
326 Positive and negative samples should be characterized using reference standard for Dengue 327 IgG (and one additional marker of Dengue - NS1 or IgM or PCR) AND IgM. The following 328 kits should be used for characterization of the sample panel: 329 • Panbio Dengue IgG capture ELISA kit 330 • WHO Pre-Qualified/ US-FDA/ ATAGI Australia/ PMDA Japan approved Dengue 331 IgM ELISA kit 332 • NS1 antigen status to be assessed using WHO Pre-Qualified/ US-FDA/ ATAGI 333 Australia/ PMDA Japan approved NS1 ELISA kit 334 • Serotype status to be assessed using a combination of CDC/NIV real-time PCR 335 serotyping protocols.
336 Sample size and sample panel composition: Sample sizes of positive and negative samples 337 of Dengue against different values of sensitivity and specificity are provided in Tables 1 and 338 2. Sample sizes have been calculated assuming 95% level of significance, an absolute precision 339 of 5%, and invalid test rate ≤5% using the following formulae: 340

341 𝑑2 x (1 −IR)
342 𝑑2 x (1 −IR)
343

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 15 | Page

344

345 346 347 348 349 350 351 352 353 Appropriate sample size has to be chosen from the tables according to the values of 354 sensitivity and specificity being claimed by the manufacturer. If a claimed 355 sensitivity/specificity is not present in the table, the manufacturer needs to consider the 356 sample size associated with the largest sensitivity/specificity provided in the table that is 357 smaller to the claimed value (that is, as per the next smaller value of the sensitivity/ 358 specificity available in the table). For example, if a manufacturer claims a sensitivity of 93%, 359 they are required to use a sample size mentioned against 90% sensitivity. Similarly, a claim 360 of 87% specificity would require usage of the sample size outlined for 85% specificity. 361

362 Positive samples: The samples should be positive for dengue IgM antibodies. The panel of 363 positive samples should include 50% of samples positive for IgG by the reference assay. 364 Samples should be representative of varying degrees of positivity: 365

366 Negative samples: These should include samples negative by all the reference assays (True 367 negatives). 368

369 Table 1. Sample sizes and panel composition of positive Dengue samples for different values 370 of sensitivity claimed by the manufacturer. 371 Sensitivity Calculated sample size Minimum no. of Positive Samples required [Sample size rounded off for balanced allocation] # Sample Panel Composition 99% 16 20

10 samples positive for Dengue IgM • Strong positive:3 • Moderate positive: 3

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• Weak positive: 4 2. 10 samples positive for both Dengue IgM and IgG • Strong positive IgG:3 • Moderate positive IgG: 3 • Weak positive IgG: 4 95% 77 80 40 samples positive for Dengue IgM • Strong positive:12 • Moderate positive: 14 • Weak positive: 14

40 samples positive for both Dengue IgM and IgG • Strong positive IgG:12 • Moderate positive IgG: 14 • Weak positive IgG: 14 90% 145 150 75 samples positive for Dengue IgM • Strong positive:23 • Moderate positive: 26 • Weak positive: 26

75 samples positive for both Dengue IgM and IgG • Strong positive IgG: 23 • Moderate positive IgG: 26 • Weak positive IgG: 26 85% 206 210 105 samples positive for Dengue IgM • Strong positive:31 • Moderate positive: 37 • Weak positive: 37

105 samples positive for both Dengue IgM and IgG • Strong positive IgG: 31 • Moderate positive IgG: 37 • Weak positive IgG: 37 80% 258 260 130 samples positive for Dengue IgM • Strong positive:38 • Moderate positive: 46 • Weak positive: 46

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130 samples positive for both Dengue IgM and IgG • Strong positive IgG: 38 • Moderate positive IgG: 46 • Weak positive IgG: 46 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity 372 373 374 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 375 specificity claimed by the manufacturer. 376 Specificity Calculated sample size Minimum no. of Negative Samples required [Sample size rounded off for balanced allocation] # Sample Panel Composition 99% 16 20 1.aSamples from acute febrile illness cases negative for dengue: 9

• Samples positive for chikungunya: 2
• Other Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR):7

2.Samples from other flavivirus disease cases (cross-reactive panel): 3 • Japanese Encephalitis IgM/IgG positive: 1@ • West Nile Virus IgM/IgG positive:1* • Zika Virus IgM/IgG positive: 1 *

bHealthy subjects from endemic regions: 8
95% 77 80 1.aSamples from acute febrile illness cases negative for dengue: 44

• Samples positive for chikungunya: 8
• Other Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR):36

2.Samples from other flavivirus disease cases (cross-reactive panel): 6

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 18 | Page

positive: 2@ • West Nile Virus IgM/IgG positive:2* • Zika Virus IgM/IgG positive: 2 *

bHealthy subjects from endemic regions: 30 90% 145 150 1.aSamples from acute febrile illness cases negative for dengue: 80

• Samples positive for chikungunya: 15
• Other Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR):65

2.Samples from other flavivirus disease cases (cross-reactive panel): 15 positive: 5 @ • West Nile Virus IgM/IgG positive:5* • Zika Virus IgM/IgG positive: 5*

bHealthy subjects from endemic regions: 55 85% 206 210 1.aSamples from acute febrile illness cases negative for dengue: 110

• Samples positive for chikungunya: 21
• Other Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR):89

2.Samples from other flavivirus disease cases (cross-reactive panel): 24 positive: 8 @ • West Nile Virus IgM/IgG positive:8* • Zika Virus IgM/IgG positive: 8*

bHealthy subjects from endemic regions: 76 80% 258 260 1.aSamples from acute febrile illness cases negative for dengue: 138

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• Samples positive for chikungunya: 26 • Other Acute febrile cases negative for Dengue (NS1 & IgM & IgG & PCR):112

2.Samples from other flavivirus disease cases (cross-reactive panel): 27 positive: 9 @ • West Nile Virus IgM/IgG positive:9* • Zika Virus IgM/IgG positive: 9*

bHealthy subjects from endemic regions: 95 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)

Cross reactivity panel is arranged in descending order of priority. The pathogens marked @ are essentially to be tested. It is recommended to test for all pathogens listed in the cross reactivity panel. However, if there is an acute shortfall or non-availability of clinical samples, one may consider reducing only the pathogens of lower priority marked by * , while ensuring that the actual numbers of cross reactive sample panel remain the same by compensating with the available “essentially to be tested” samples.

Note: If IgM/IgG positive samples for cross reactive flaviviruses are not available, commercially available IgM/IgG sera panel for different viruses can be procured and used to test cross reactivity.

377

378 5. Evaluation method: 379 The index test and the reference assay should be run simultaneously on the sample panel, 380 and results should be recorded.
381

382 6. Resolution of discrepant results: 383 True positive samples: These are samples positive by reference assay and index test. 384 True negative samples: These are samples negative by reference assay and index test. 385

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386 index test. 387 388 index test. 389

390 7. Test reproducibility 391 C. Sample size for lot-to-lot reproducibility 392 Three lots of an assay shall be evaluated. Sample size for lot-to-lot reproducibility should be 393 as follows:
394 395 and negative samples as calculated in the protocol.
396 • Second lot of the assay: should be tested on 25 samples (15 positive samples 397 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 398 samples).
399 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 400 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 401

402 403

404

405

406 D. Sample size for reader-to-reader reproducibility 407

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For reader-to-reader reproducibility, sample size should be 25 (15 positive samples comprising 10 408 low positive AND 5 moderate/high positive samples, and 10 negative samples). 409

410 Two operators will be reading the test results independently as per manufacturer’s instruction. 411 Agreement should be 100% between the operators. 412 8. Blinding of laboratory staff 413 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should 414 be blinded to the status of the clinical samples. The PI of the evaluation exercise should 415 remain unblinded, i.e., privy to the status of the samples. Another senior laboratory staff 416 selected by the PI may remain unblinded and carry out coding of samples and dispensing 417 them into similar-looking vials to be used for testing, and maintaining the database of 418 results. Staff performing the reference test and the test under evaluation, interpretation of 419 the test result, and entering the results against the coded samples in the database, should 420 remain blinded to the status of samples till the completion of evaluation. The data should 421 be analyzed only by the PI of the evaluating lab. Refer to Fig. 2. 422

423 424

425

426 9. Acceptance Criteria
427 Expected sensitivity for each analyte: ≥80% 428 Expected specificity for each analyte: ≥90% 429

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430 431 To achieve at least the performance characteristics outlined in the acceptance criteria, ≥260 432 positive samples and ≥150 negative samples should be used for evaluation. 433 10. Publication Rights: 434 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead author(s). 435

436 437 438 acceptable.
439 440 441 442 443 444 445 446 447

448 VI. References: 449

Vazquez S, Hafner G, Ruiz D, Calzada N, Guzman MG. Evaluation of immunoglobulin M and G 450 capture enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue infections. J Clin Virol. 451 2007 Jul;39(3):194-8. doi: 10.1016/j.jcv.2007.04.003..
452
WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. (Diagnostics 453 evaluation series, 3). ISBN 978 92 4 159775 3.
454
Central Drugs Standard Control Organization. Guidance on Performance Evaluation of In-vitro 455 Diagnostic Medical Devices.

Available at: 456 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/medical 457 device/guidanceperformanceivd.pdf 458 4. Central Drugs Standard Control Organization. In-Vitro Diagnostic (IVD) Medical Devices Frequently 459 Asked Questions. 2022. Available at: https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf- 460 documents/IVD/FAQs/CDSCO-IVD-FAQ-03-2022-.pdf
461 5. U.S. Food and Drug Administration. Dengue Virus Serological Reagents - Class II Special Controls 462 Guideline for Industry and Food and Drug Administration Staff. 2014. Available at: 463 https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation-emitting- 464 products/Dengue-virus-serological-reagents-class-ii-special-controls-guideline-industry-and-food- 465 and-drug
466

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World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – Diagnostic 467 Assessment TGS-3. 2017. Available at: https://iris.who.int/bitstream/handle/10665/258985/WHO- 468 EMP-RHT-PQT-TGS3-2017.03-eng.pdf;sequence=1
469

470 *The validation protocols need to be revisited after introduction of Dengue vaccines and the 471 acceptance criteria needs revisiting every year so as to enable the availability of best 472 diagnostic kits.
473

474 VII. Performance evaluation report format 475

476

477

478

479

480

481

482

483

484

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494

495

496

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PERFORMANCE EVALUATION REPORT FOR DENGUE IgM and IgG COMBO RDT 497 KIT 498

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

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

500 Results for IgM: 501

Reference assay ……….……………… (name)

Positive Negative Total Name of Dengue antibody combo RDT kit Positive

Negative

Total

502 Results for IgG: 503

Reference assay ……….……………… (name)

Positive Negative Total

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 25 | Page

Name of Dengue antibody combo RDT kit Positive

Negative

Total

504

505 ● Details of cross reactivity with other flavivirus antibodies:
506 ● Invalid test rate: 507

508 ● Conclusions: 509 o Sensitivity, specificity for dengue IgM: 510 o Sensitivity, specificity for dengue IgG: 511 o Performance:
512 ▪ Satisfactory / Not satisfactory for Dengue IgM 513 ▪ Satisfactory / Not satisfactory for Dengue IgG 514

515 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from 516 the batch mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 517 Disclaimers 518 519 520

521 Note: This report is exclusively for ………….. Kit (Lot No……) manufactured by …………… (Supplied by ……….) 522 523 524 Signature of Director/ Director-In-charge …………………… Seal ……………………………… 525 ****End of the Report 526

527

528

529

530

531

532

533

534

535

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 26 | Page

Performance evaluation protocol for Dengue IgG ELISA kits 536 I. Background:
537 CDSCO/ICMR, New Delhi, have aimed at facilitating the availability of Quality-Assured 538 Diagnostics kits appropriate for use in India. Hence the following guidelines shall establish the 539 uniformity in performance evaluation of in-vitro diagnostic kits (IVD). The performance 540 evaluation is to independently verify the manufacturer’s claim regarding in-vitro diagnostic kit 541 (IVD) performance. 542 II. Purpose: 543 To evaluate the performance characteristics of Dengue IgG ELISA kits in the diagnosis of Dengue 544 infection using irreversibly de-identified leftover archived clinical samples. 545 III. Requirements:
546

Supply of kits under evaluation (Along with batch/lot No. Expiry & required details). If 547 the kit to be evaluated works in a closed system format, the manufacturer needs to supply 548 the required equipment. 549
Evaluation sites/laboratories (With required equipment) 550
Reference test kits 551
Characterised Evaluation panel 552
Laboratory supplies
553 IV. Ethical approval:
554 Performance evaluation activities using irreversibly de-identified leftover clinical samples are 555 exempt from ethics approval as per ICMR’s Guidance on Ethical Requirements for Laboratory 556 Validation Testing, 2024.
557 Investigators are required to submit a self-declaration form, as outlined in the ICMR guidelines, 558 to the institutional authorities and ethics committee for information. 559 V. Procedure: 560
Study design/type: Diagnostic accuracy study using irreversibly de-identified archived/ 561 spiked leftover clinical samples 562 563 Identified IVD kit evaluation laboratories should establish their proficiency through 564 ALL of the following: 565 A. Accreditation for at least one of the Quality management systems (accreditation for Testing 566 Lab / Calibration Lab (ISO: 17025), Medical Lab (ISO: 15189), PT provider (ISO: 17043) or 567 CDSCO approved Reference laboratory. 568

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B. Staff training: All the staff involved in IVD kit evaluation should undergo hands on training 569 and competency testing on following
570 ⮚ Preparation & characterization of kit evaluation panel
571 ⮚ Handling of Dengue IgG ELISA IVD kits received for performance evaluation 572 (Verification/Storage/Unpacking etc). 573 ⮚ Testing, interpreting, recording of results & reporting 574 ⮚ Data handling, data safety & confidentiality 575 3. Preparation of Dengue IgG ELISA IVD kit evaluation panel 576 Well characterised Dengue IVD kit evaluation panel is a critical requirement for performance 577 evaluation of IVD kits. Hence statistically significant number of sera samples should be 578 collected from Dengue NS1/PCR/IgG confirmed cases. Further characterised for Dengue IgM 579 positivity by using approved reference kits having high sensitivity and specificity. 580 Dengue IgG performance evaluation panel need to be tested again by the reference assays at 581 the time of evaluating a particular index test to confirm the positive and negative status of the 582 samples. 583 4. Reference assay:
584 Positive and negative samples should be characterized using composite reference standard 585 of Dengue IgG AND one additional marker of Dengue (NS1 or IgM or PCR). The 586 following kits should be used for characterization of the sample panel: 587 • Panbio Dengue IgG capture ELISA kit 588 • WHO Pre-Qualified/ US-FDA/ ATAGI Australia/ PMDA Japan approved Dengue 589 IgM ELISA kit 590 • NS1 antigen status to be assessed using WHO Pre-Qualified/ US-FDA/ ATAGI 591 Australia/ PMDA Japan approved NS1 ELISA kit 592 • Serotype status to be assessed using a combination of CDC/NIV real-time PCR 593 serotyping protocols.
594

595 5. Sample size for performance evaluation: 596 Sample sizes of positive and negative samples and sample panel composition against different 597 values of sensitivity and specificity are provided in Tables 1 and 2. Sample sizes have been 598 calculated assuming 95% level of significance, and an absolute precision of 5% using the 599 following formulae: 600

601 𝑑2

602

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 28 | Page

𝑑2

603

604

605 606 607 608 609 610 611 612 ·
613 Appropriate sample size has to be chosen from the tables according to the values of sensitivity 614 and specificity being claimed by the manufacturer. If a claimed sensitivity/specificity is not 615 present in the table, the manufacturer needs to consider the sample size associated with the 616 largest sensitivity/specificity provided in the table that is smaller to the claimed value (that is, 617 as per the next smaller value of the sensitivity/ specificity available in the table). For example, 618 if a manufacturer claims a sensitivity of 93%, they are required to use a sample size mentioned 619 against 90% sensitivity. Similarly, a claim of 87% specificity would require usage of the 620 sample size outlined for 85% specificity.
621

622 Positive samples: The panel of positive samples should include samples positive for IgG by the 623 reference assay. The samples should also be positive for either dengue NS1 antigen or dengue IgM 624 antibodies. Samples should be representative of varying degrees of positivity: 625

626 Negative samples: These should include samples negative by the reference assays for dengue IgG.
627

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 Minimum no. of Positive Samples required [Sample size rounded off] # Sample Panel Composition 99% 15 20 Strong Positive: 6 Moderate Positive: 7 Weak Positive: 7 95% 73 80 Strong Positive: 24 Moderate Positive: 28

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 29 | Page

Weak Positive: 28 90% 138 140 Strong Positive: 42 Moderate Positive: 49 Weak Positive: 49 85% 196 200 Strong Positive: 60 Moderate Positive: 70 Weak Positive: 70 80% 246 250 Strong Positive: 75 Moderate Positive: 87 Weak Positive: 88

631 #It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and specificity; 632 however, a higher sample size is suggested to ensure adequate power of the study in case the kit falls short 633 of claimed performance characteristics. 634 Table 2. Sample sizes and panel composition of negative Dengue samples for different values of 635 specificity claimed by the manufacturer. 636 Specificity Calculated sample size Minimum no. of Negative Samples required [Sample size rounded off]# Sample Panel Composition 99% 15 20 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 7
2.Acute febrile illness cases: 8
• Chikungunya positive samples:2 negative samples:6 3.Samples from other flavivirus disease cases (cross-reactive panel): 3 positive: 1 @ • West Nile Virus IgM/IgG positive: 1 * • Zika Virus IgM/IgG positive: 1 * 4. aHealthy subjects from endemic regions: 2
95% 73 80 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 27
2.Acute febrile illness cases: 32
• Chikungunya positive samples:8 negative samples:24

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 30 | Page

3.Samples from other flavivirus disease cases(cross-reactive panel): 9 positive: 3 @ • West Nile Virus IgM/IgG positive: 3 * • Zika Virus IgM/IgG positive: 3 * 4. aHealthy subjects from endemic regions: 12
90% 138 140 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 45
2.Acute febrile illness cases: 60
• Chikungunya positive samples:15 negative samples:45 3.Samples from other flavivirus disease cases(cross-reactive panel): 15 positive: 5 @ • West Nile Virus IgM/IgG positive: 5 * • Zika Virus IgM/IgG positive: 5 * 4. aHealthy subjects from endemic regions: 20
85% 196 200 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 65
2.Acute febrile illness cases: 84
• Chikungunya positive samples:21 negative samples:63 3.Samples from other flavivirus disease cases(cross-reactive panel): 21 positive: 7 @ • West Nile Virus IgM/IgG positive: 7 * • Zika Virus IgM/IgG positive: 7 * 4. aHealthy subjects from endemic regions: 30
80% 246 250 1.Samples positive for dengue IgM/NS1/RNA but negative for IgG: 80
2.Acute febrile illness cases: 104
• Chikungunya positive samples:26 negative samples:78 3.Samples from other flavivirus disease cases(cross-reactive panel): 27 positive: 9 @ • West Nile Virus IgM/IgG positive: 9 * • Zika Virus IgM/IgG positive: 9 *

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 31 | Page

aHealthy subjects from endemic regions: 39
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)

#It is recommended to calculate the sample size as per manufacturer's claims of sensitivity and specificity; however, a higher sample size is suggested to ensure adequate power of the study in case the kit falls short of claimed performance characteristics. Cross reactivity panel is arranged in descending order of priority. The pathogens marked @ are essentially to be tested. It is recommended to test for all pathogens listed in the cross reactivity panel. However, if there is an acute shortfall or non-availability of clinical samples, one may consider reducing only the pathogens of lower priority marked by * , while ensuring that the actual numbers of cross reactive sample panel remain the same by compensating with the available “essentially to be tested” samples.

Note: If IgM/IgG positive samples for cross reactive flaviviruses are not available, commercially available IgM/IgG sera panel for different viruses can be procured and used to test cross reactivity.

637

638

639 6. Evaluation method: 640 The index test and the reference assay should be run simultaneously on the sample panel, 641 and results should be recorded.
642

643 7. Interpretation of results:
644 Reference test and index test results will be interpreted as per kit IFU.
645 8. Resolution of discrepant results: 646 True positive samples: These are samples positive by reference assay and index test. 647 True negative samples: These are samples negative by reference assay and index test. 648 649 index test. 650 651 index test. 652

653 9. Test reproducibility 654 A. Sample size for lot-to-lot reproducibility 655 Three lots of an assay shall be evaluated. Sample size for lot-to-lot reproducibility should be 656 as follows:
657

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 32 | Page

658 and negative samples as calculated in the protocol.
659 • Second lot of the assay: should be tested on 25 samples (15 positive samples 660 comprising 10 low positive AND 5 moderate/high positive samples, and 10 negative 661 samples).
662 • Third lot of the assay: should be tested on 25 samples (15 positive samples comprising 663 10 low positive AND 5 moderate/high positive samples, and 10 negative samples). 664

665 666

667

668

669

670 10. Blinding of laboratory staff 671 To ensure rigor of the evaluation process, laboratory staff performing the evaluation should 672 be blinded to the status of the clinical samples. The PI of the evaluation exercise should 673 remain unblinded, i.e., privy to the status of the samples. Another senior laboratory staff 674 selected by the PI may remain unblinded and carry out coding of samples and dispensing 675 them into similar-looking vials to be used for testing, and maintaining the database of 676 results. Staff performing the reference test and the test under evaluation, interpretation of 677 the test result, and entering the results against the coded samples in the database, should 678 remain blinded to the status of samples till the completion of evaluation. The data should 679 be analyzed only by the PI of the evaluating lab. Refer to Fig. 2. 680

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 33 | Page

681 682

683

684

685 11. Acceptance criteria 686 Expected sensitivity: ≥90% 687 Expected specificity: ≥95%
688 689 To achieve at least the performance characteristics outlined in the acceptance criteria, 690 ≥140 positive samples and ≥80 negative samples should be used for evaluation. 691 12. Publication Rights: 692 The PI(s) of the evaluating labs shall retain publication rights of the evaluation as lead 693 author(s). 694

695 696 697 acceptable.
698

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 34 | Page

699 700 701 702 703 704 705 706

707 VI. References: 708

Vazquez S, Hafner G, Ruiz D, Calzada N, Guzman MG. Evaluation of immunoglobulin M 709 and G capture enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue 710 infections. J Clin Virol. 2007 Jul;39(3):194-8. doi: 10.1016/j.jcv.2007.04.003..
711
WHO, Evaluation of commercially available anti-Dengue virus immunoglobulin M tests. 712 (Diagnostics evaluation series, 3). ISBN 978 92 4 159775 3.
713
Central Drugs Standard Control Organization. Guidance on Performance Evaluation of In-vitro 714 Diagnostic Medical Devices.

Available at: 715 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/medical 716 device/guidanceperformanceivd.pdf 717 4. Central Drugs Standard Control Organization. In-Vitro Diagnostic (IVD) Medical Devices 718 Frequently Asked Questions. 2022. Available at: 719 https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/IVD/FAQs/CDSCO- 720 IVD-FAQ-03-2022-.pdf
721 5. U.S. Food and Drug Administration. Dengue Virus Serological Reagents - Class II Special Controls 722 Guideline for Industry and Food and Drug Administration Staff. 2014. Available at: 723 https://www.fda.gov/medical-devices/guidance-documents-medical-devices-and-radiation- 724 emitting-products/Dengue-virus-serological-reagents-class-ii-special-controls-guideline-industry- 725 and-food-and-drug
726 6. World Health Organization. Technical Guidance Series (TGS) for WHO Prequalification – 727 Diagnostic Assessment TGS-3. 2017. Available at: 728 https://iris.who.int/bitstream/handle/10665/258985/WHO-EMP-RHT-PQT-TGS3-2017.03- 729 eng.pdf;sequence=1
730

731 *The validation protocols need to be revisited after introduction of Dengue vaccines and the 732 acceptance criteria needs revisiting every year so as to enable the availability of best 733 diagnostic kits.
734

735 VII. Performance evaluation report format 736

737

738

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 35 | Page

PERFORMANCE EVALUATION REPORT FOR DENGUE IgG ELISA KIT 739 Lot No / Batch No.: Type of Assay Kit components Manufacturing Date Expiry Date Intended Use

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

740 Results:
741

Reference assay ……….……………… (name)

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

Negative

Total

742

Estimate (%) 95% CI Sensitivity

Specificity

Conclusions: 743 o Sensitivity, specificity 744 o Cross-reactivity: 745 o Invalid test rate: 746 o Performance: Satisfactory / Not satisfactory 747 (Sensitivity and specificity have been assessed in controlled lab setting using kits provided by the manufacturer from the batch 748 mentioned above using ….. sample. Results should not be extrapolated to other sample types.) 749

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 36 | Page

Disclaimers 750 751 752 Note: This report is exclusively for ………………………Kit (Lot No……) manufactured by …………… (Supplied 753 by ……….) 754 755 756 Signature of Director/ Director-In-charge …………………… Seal……………………………………… 757

758 ****End of the Report 759

760

761

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ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 37 | Page

Information on Operational and Test Performance Characteristics Required from Manufacturers 781 for Dengue IgG Based Assays 782 The manufacturer should provide the following details about the IVD: 783

Instructions for Use 784
Scope of the IVD: to diagnose Dengue
785
Intended Use Statement 786
Principle of the assay 787
Intended testing population(cases of acute febrile illness/suspected cases of Dengue) 788
Intended user(laboratory professional and/or health care worker at point-of-care) 789
Detailed test protocol 790
Lot/batch No. 791
Date of manufacture 792
Date of Expiry 793
Information on operational Characteristics 794 i. Configuration of the kit/device 795 ii. Requirement of any additional equipment, device 796 iii. Requirement of any additional reagents 797 iv. Operation conditions 798 v. Storage and stability before and after opening 799 vi. Internal control provided or not 800 vii. Quality control and batch testing data 801 viii. Biosafety aspects- waste disposal requirements 802
Information on Test Performance Characteristics 803 i. Type of sample-serum/plasma/whole blood/other specimen (specify) 804 ii. Volume of sample 805 iii. Sample rejection criteria (if any) 806 iv. Any additional sample processing required 807 v. Any additional device/consumable like sample transfer device, pipette, tube, etc required 808 vi. Name of analyte to be detected 809 vii. Pathogens targeted by the kit 810

ICMR-CDSCO/IVD/GD/PROTOCOLS/10/2025 38 | Page

viii. Time taken for testing 811 ix. Time for result reading and interpretation 812 x. Manual or automated(equipment)reading 813 xi. Limit of detection 814 xii. Diagnostic sensitivity 815 xiii. Diagnostic specificity 816 xiv. Stability and reproducibility (including data) 817 xv. Training required for testing (if any) 818 xvi. If yes, duration 819 xvii. Details of Cut-off and /or Equivocal Zone for interpretation of test 820 xviii. Details of cross reactivity, if any 821 xix. Interpretation of invalid and indeterminate results to be provided 822 xx. It is recommended to provide data demonstrating the precision 823

824 *Please mention “Not applicable” against sections not pertaining to the kit. 825

826

827 ****End of the Document 828

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