Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 5 (2013)

MalariaRapid Rapid Diagnostic Malaria Diagnostic Test Performance Performance Test Results of WHO product testing of Results of WHO product testing of malaria RDTs: Round 4 (2012) malaria RDTs: Round 5 (2013)

Malaria Rapid Diagnostic Test Performance Results of WHO product testing of malaria RDTs: Round 5 (2013)

Reference to any company or product in this report, particularly those listed in any of the figures and tables, does not constitute an endorsement, certification, or warranty of fitness by WHO of such company or product for any purpose, and does not imply any preference over companies or products of a similar nature that are not mentioned. WHO does not furthermore warrant that: (1) the lists and figures are complete and/or error free; and/or that (2) any products included in the figures and tables are of acceptable quality, have obtained regulatory approval in any country, or that their use is otherwise in accordance with the national laws and regulations of any country, including but not limited to patent laws. Inclusion of any products in this report, particularly in any of the figures and tables listed on pages V-VII, does not furthermore imply any approval by WHO of these products (which is the sole prerogative of national authorities). The WHO Programme of Prequalification of Diagnostics and Medical Devices uses the results of the WHO Malaria RDT Product Testing Programme as the laboratory evaluation component of the prequalification process for malaria RDTs. Although not currently a requirement for WHO procurement, manufacturers are encouraged to apply for WHO prequalification. A regularly updated list of WHO-prequalified diagnostics, including malaria RDTs, is available at http://www.who.int/diagnostics_laboratory/evaluations/PQ_list/en/. WHO recommendations for procurement of malaria RDTs are currently based on the attainment of a set of minimum performance criteria in the WHO Malaria RDT Product Testing Programme. These recommendations were established by the WHO Malaria Policy Advisory Committee in 2012 , are outlined in this report and presented in full in a WHO information note (available at http://www.who.int/malaria/ publications/atoz/rdt_selection_criteria_en.pdf?ua=1).Products that do not meet the full set of minimum performance criteria are not eligible for procurement by WHO. The lists of RDTs included in this report are not exhaustive lists of malaria RDTs. These lists reflect those products which have been submitted for evaluation in Rounds 2-5 of the WHO Malaria RDT Product Testing Programme, and indicate to what extent these products, as manufactured by the listed companies, were -at the time of their evaluation- found to meet the above mentioned set of minimum performance criteria. The evaluation results indicated in the figures and tables apply only to the specific product as listed with its unique product code / catalogue number and as manufactured by the listed company. The improper storage, transport and handling of malaria RDTs may affect their level of performance. The fact that certain products are not included in the lists and figures in this report indicates that they have not or not yet been submitted for evaluation in the WHO Malaria RDT Product Testing Programme, or that their evaluation has not yet been completed and published in [a new edition of this report]. It does not however indicate anything in respect of such products’ performance. The lists and figures are updated regularly, and malaria RDTs are added to the lists and figures as and when (following the voluntary participation in the WHO Malaria RDT Product Testing Programme) their evaluation against the above mentioned set of minimum performance criteria has been completed. Although the malaria RDTs listed in the tables and figures are regularly re-evaluated, and updated evaluation results are published by WHO, WHO cannot represent that products included in the lists and figures will continue to meet the performance criteria in the same manner as indicated. WHO recommends therefore that before procurement of a malaria RDT, each lot of that product undergoes lot testing at one of the two following lot-testing laboratories: Institut Pasteur du Cambodge (IPC), Cambodia or Research Institute for Tropical Medicine (RITM), The Philippines. WHO disclaims any and all liability and responsibility whatsoever for any injury, death, loss, damage, or other prejudice of any kind that may arise as a result of or in connection with the procurement, distribution and use of any product included in this report and the figures and tables listed on pages V-VII. This report may not be used by manufacturers and suppliers for commercial or promotional purposes.

WHO Library Cataloguing-in-Publication Data : Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 5 (2013). 1.Diagnostic Techniques and Procedures. 2.Malaria - diagnosis. 3.Diagnostic Tests, Routine - methods. I.World Health Organization. ISBN 978 92 4 150755 4 (NLM classification: WC 750)

© World Health Organization 2014 All rights reserved. Publications of the World Health Organization are available on the WHO website (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications –whether for sale or for non-commercial distribution– should be addressed to WHO Press through the WHO website (www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. Layout: Bruno Duret - Editor: Elisabeth Heseltine Printed in Italy

Contents

Acknowledgements VIII Abbreviations IX 1. SUMMARY OF PERFORMANCE OF RAPID DIAGNOSTIC TESTS FOR MALARIA: WHO PRODUCT TESTING ROUNDS 1–5 1.1. Introduction 1.2. The WHO product testing programme 1.3.  Panel detection score and other results of the evaluation 1.4. Summary of outcomes 1.5.  How can product testing results inform RDT procurement and use? 1.6.  Product testing and WHO programme for prequalification of diagnostics and medical devices

1 1 1 2 4 5 5

2. WHO MALARIA RDT PRODUCT TESTING: Round 5: Executive Summary 2.1. Introduction 2.2. The WHO product testing programme 2.3. Results of the evaluation 2.4.  Use of the results

20 20 20 21 22

3. Background 4. Objective 5. Materials and methods 5.1. Test selection 5.2. The product testing protocol 5.3. Evaluation panels 5.4. RDT registration 5.5. Specimen panel registration 5.6. Test phases 5.7.  Performing rapid tests 5.8.  Interpreting the results

22 24 24 24 26 27 29 29 29 29 30

6. Data management 7.  Quality assurance 8. Ethical considerations 9. Data analysis 9.1. Measures of parasite detection: panel detection score and positivity rates 9.2.  False-positive results 9.2.1  Incorrect species identification 9.2.2  False-positive results for Plasmodium-negative samples

31 31 32 32 32 32 32 33

9.3. Band intensity 9.4. Lot agreement 9.5.  Invalid tests 9.6.  Heat (thermal) stability

33 33 33 33

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

III

10.   RELATION BETWEEN PARASITE DENSITY AND ANTIGEN CONCENTRATION 11.   Laboratory versus field-based malaria RDT evaluations 12. Results 12.1. Summary 12.2.  Phase 1: P. falciparum culture panel 12.3.1  P. falciparum detection 12.3.2  P. vivax detection 12.3.3 Combined detection of P. falciparum and P. vivax P. falciparum and P. vivax positivity rate 12.3.4   12.3.5 Band intensity 12.3.6  False-positive rates

34 34 35 35 40 41 42 42 43 43 45

12.4.  Performance of products under the compulsory resubmission requirement

48

13.  Heat stability

50

Ease-of-use DESCRIPTION and RDT 14.   anomalies 57 15. Discussion of key findings 15.1.  Panel detection score and its relation to sensitivity 15.2.  False-positive rate and specificity 15.3. Reactivity of combination HRP2 and pan-pLDH test lines against P. falciparum samples 15.4.  Heat (thermal) stability 15.5. Ease-of-use description and RDT anomalies in production lots 15.6.  Inter-lot variation 15.7. Target antigens and species

60 60 61 61 62 62 63 63

16.  USING RESULTS TO ENSURE HIGH-QUALITY DIAGNOSIS IN THE FIELD 64 16.1. Beyond procurement 16.2. Lot testing 64 64

17. Conclusions 18. References

65 65

Annexes 67 Annex S1: Characteristics of evaluation panels used in rounds 1–5 of WHO malaria RDT product testing, 2008–2013 68 Annex S2: Malaria RDT field assessment and anomalies 71 Annex S3: Selection of an appropriate RDT 74 Annex 1: Characteristics of RDTs evaluated in round 5 75 Annex 2: Malaria RDTs: guide to interpretation of results 77 Annex 3: Phase-1 results 92 Annex 4: Phase-2 results 96 Annex 5:  Introducing RDT-based malaria diagnosis into national programmes 125

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

figures Figure S1: Figure S2: Figure S3: Malaria RDT performance in phase 2 of rounds 2–5 against wild-type (clinical) samples containing P. falciparum at low (200) and high (2000 or 5000) parasite density (parasites/µL) and clean-negative samples Malaria RDT performance in phase 2 of rounds 2–5 against wild-type (clinical) samples containing P. vivax at low (200) and high (2000 or 5000) parasite density (parasites/µL) and clean-negative samples Panel detection score of malaria combination RDTs meeting WHO procurement criteria for false-positive and invalid rates, in phase 2 of rounds 2–5 against wild-type (clinical) samples containing P. falciparum and P. vivax at low parasite density (200 parasites/μL) Mode of action of antigen-detecting malaria RDTs Network of specimen collection, characterization and testing sites Overview of malaria RDT product testing Origin of phase-2 P. falciparum wild-type (clinical) samples Origin of phase-2 P. vivax wild-type (clinical) samples Testing procedure and calculation of panel detection score and band intensity for product A against a sample density of 200 parasites/μL Testing procedure and calculation of panel detection score and band intensity for product A against a sample density of 2000 parasites/μL Classification of incorrect species identification with combination malaria RDTs Explanation of lot agreement calculation

Figure 1: Figure 2: Figure 3: Figure 4a: Figure 4b: Figure 5: Figure 6: Figure 7: Figure 8:

Figure 9: Phase-1 P. falciparum panel detection score of malaria RDTs at low (200) and high (2000) parasite density (parasites/µL) according to target antigen type (HRP2 or pLDH) Figure 10: Phase-2 P. falciparum panel detection score of malaria RDTs at low (200) and high (2000) parasite density (parasites/µL) according to target antigen type (HRP2 or pLDH) Figure 11: Phase-2 P. vivax panel detection score of malaria RDTs at low (200) and high (2000) parasite density (parasites/ µL) according to target antigen type (aldolase, pLDH) Figure 12: Phase-2 P. falciparum panel detection score and positivity rate at 200 parasites/μL Figure 13: Phase-2 P. vivax panel detection score and positivity rate at 200 parasites/μL Figure 14: Phase-2 P. falciparum (P. falciparum test line) false-positive rate against clean-negative samples Figure 15: Phase-2 Plasmodium spp. (pan or P. vivax/Pvom test line) false-positive rate against clean-negative samples Figure 16: Phase-2 P. falciparum false-positive rate versus P. falciparum panel detection score at low parasite density (200 parasites/μL) Figure 17: Phase-2 P. vivax false-positive rate versus P. vivax panel detection score at low parasite density (200 parasites/μL) Figure 18: Phase-2 P. falciparum panel detection score at low parasite density (200 parasites/μL) during initial and subsequent testing of compulsorily and voluntarily resubmitted malaria RDTs Figure 19: Phase-2 P. vivax panel detection score at low parasite density (200 parasites/μL) during initial and subsequent testing of compulsorily and voluntarily resubmitted malaria RDTs Figure 20: Figure 21: Figure 22: Figure 23: Figure 24: Heat stability of P. falciparum-specific test line of P. falciparum- only tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation Heat stability of P. falciparum-specific test line of P. falciparum-only tests against a high-density P. falciparum sample (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation Heat stability of P. falciparum -specific test line in combination tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation Heat stability of P. falciparum -specific test line in combination tests against a high-density P. falciparum sample (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation Heat stability of pan line of pan-specific tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation

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V

Figure 25: Figure 26: Figure 27:

Heat stability of pan line of pan-specific tests against a high-density P. falciparum sample (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation Heat stability of pan line of combination tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation Heat stability of pan line of combination tests against a high-density P. falciparum sample (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation

Figure AS1.1: Box-and-whisker plot of distribution of P. falciparum HRP2 concentration (ng/mL) in product testing phase 2 (wild-type) panels Figure AS1.2: Box-and-whisker plot of distribution of P. falciparum pLDH concentration (ng/mL) in product testing phase 2 (wild-type) panels Figure AS1.3: Box-and-whisker plot of distribution of P. vivax pLDH concentration (ng/mL) in product testing phase 2 (wild-type) panels Figure AS1.4: Box-and-whisker plot of distribution of P. falciparum aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels Figure AS1.5: Box-and-whisker plot of distribution of P. vivax aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels Figure AS2.1: Malaria RDT anomalies encountered in production lots Figure AS3.1: Selecting an appropriate RDT Figure A5.1: Example of malaria RDT implementation steps and timeline Figure A5.2: Components of the budget for a malaria diagnosis programme

Tables Table S1: Table S2 Table S3: Table 1a: Table 1b: Table 2: Table 3: Table 4: Table 5: Table 6: Product resubmissions: WHO malaria RDT product testing rounds 1–5 Malaria RDT phase-2 performance in rounds 2–5 against wild-type (clinical) samples containing P. falciparum and P. vivax at low (200) and high (2000 or 5000) parasite density (parasites/µL) and clean-negative samples Malaria RDT rounds 2–5 heat stability results on a cultured P. falciparum sample at low (200) and high (2000) parasite density (parasites/µL). Positivity rate at baseline and after 60 days’ incubation at 35 °C and 45 °C Manufacturers and products accepted into round 5 of WHO malaria RDT product testing programme Products due for compulsory resubmission in round 5 Characteristics of Plasmodium spp.-negative samples Malaria antigen concentrations (ng/mL) in round 5 wild-type, low-density (200 parasites/μL) samples Summary of phase-1 performance of 42 malaria RDTs against 20 cultured P. falciparum lines at low (200) and high (2000) parasite density (parasites/µL) Summary of phase-2 performance of 42 malaria RDTs against wild-type (clinical) P. falciparum and P. vivax samples at low (200) and high (2000) parasite density (parasites/µL) and Plasmodium spp.-negative samples Heat stability testing results for 42 malaria RDTs on a cultured P. falciparum sample at low (200) and high (2000) parasite density (parasites/µL). Positivity rate at baseline (room temperature) and after 60 days’ incubation at 35 °C and 45 °C Ease-of-use description of 42 malaria RDTs evaluated in round 5 of WHO malaria RDT product testing Observations on RDT production lots that could affect test interpretation

Table 7: Table 8:

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Table AS1.1: Statistics for P. falciparum HRP2 concentration (ng/mL) in product testing phase 2 (wild-type) panels Table AS1.2: Statistics for P. falciparum pLDH concentration (ng/mL) in product testing phase 2 (wild-type) panels Table AS1.3: Statistics for P. vivax pLDH concentration (ng/mL) in wild-type product testing phase 2 (wild-type) panels. Table AS1.4: Statistics for P. falciparum aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels Table AS1.5: Statistics for P. vivax aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels Table AS2.1: Field assessment of RDT packaging, safety and ease-of-use to guide product selection Table A3.1: Table A3.2: Table A4.1: Table A4.2: Table A4.3: Table A4.4: Lot variation in positive results against P. falciparum culture samples at low (200) and high (2000) parasite density (parasites/µL) Distribution of test band intensity scores (0–4) against phase-1 P. falciparum cultured parasites at low (200) and high (2000) parasite density (parasites/µL) Lot variation in positive results against phase-2 wild-type P. falciparum and P. vivax samples at low (200) and high (2000) parasite density (parasites/µL) Distribution of test band intensity (0–4) scores against phase-2 wild-type P. falciparum samples at low (200) and high (2000) parasite density (parasites/µL) Distribution of pan/Pv test band intensity (0–4) scores for phase-2 wild-type P. vivax samples at low (200) and high (2000) parasite density (parasites/µL) Panel detection score of phase-2 wild-type P. falciparum at low (200) and high (2000) parasite density (parasites/µL) by continent

Table A4.5: Phase-2 P. falciparum test line false-positive rates for wild-type P. vivax samples at low (200) and high (2000) parasite density (parasites/µL) Table A4.6: Table A4.7: Table A4.8: Table A4.9: Phase-2 pan (or P. vivax/Pvom) test line false-positive rate for non-P. falciparum infection on phase-2 wildtype P. falciparum samples at low (200) and high (2000) parasite density (parasites/µL) Phase-2 false-positive rate for P. falciparum test line results on all malaria-negative samples Phase-2 false-positive rate for P. falciparum in samples containing non-malarial infectious pathogens Phase-2 false-positive rate for P. falciparum in samples containing potentially cross-reacting blood immunological factors

Table A4.10: Phase-2 false-positive rate of pan, P. vivax or Pvom test line results in all malaria-negative samples Table A4.11: Heat stability testing results for P. falciparum (or pan) test line on a P. falciparum sample at low parasite density (200 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Table A4.11a: Heat stability testing results for pan test line of combination RDTs on a P. falciparum sample at low parasite density (200 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Table A4.12: Heat stability testing results for P. falciparum (or pan) test line on a P. falciparum sample at high parasite density (2000 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Table A4.12a: Heat stability testing results for pan test line of combination RDTs on a P. falciparum sample at high parasite density (2000 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Table A4.13: Heat stability testing results for P. falciparum (or pan) test line on parasite-negative samples. Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Table A4.13a: Heat stability testing results for pan test line of combination RDTs on parasite-negative samples. Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C

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VII

Acknowledgements The evaluation reported here was a joint project of the WHO Global Malaria Programme, the Foundation for Innovative New Diagnostics (FIND) and the United States Centers for Disease Control and Prevention (CDC) within the WHO-FIND Malaria RDT Evaluation Programme. The project was financed by FIND through a grant from UNITAID. The project would not have been possible without the cooperation and support of the specimen collection sites and specimen characterization laboratories mentioned, and the authors acknowledge the technical advice from many malaria diagnostic manufacturers and developers. This report of round 5 of WHO malaria RDT product testing was compiled by Jane Cunningham (WHO, Global Malaria Programme, Switzerland) and Michelle Gatton (Queensland University of Technology, University of Queensland, Australia). The malaria RDT evaluation programme of WHO and FIND are grateful to all those who contributed to the evaluation and to the preparation of this report:

Salim Abdullah Yong Ah Frederic Ariey John Barnwell David Bell Andrea Bosman Qin Cheng Peter Chiodini Jane Cunningham Chona Daga Djibrine Djalle Babacar Faye Dionicia Gamboa Michelle Gatton Jeffrey Glenn Iveth Gonzalez Sandra Incardona Cara Kosack Myat Phone Kyaw Brigette Lawhorn Jennifer Luchavez Christian Luna Lorraine Mationg James McCarthy Didier Menard Claribel Murillo

Ifakara Health Research and Development Centre, United Republic of Tanzania United States Centers for Disease Control and Prevention, National Center for Global Health, Division of Malaria and Parasitic Diseases, United States Institut Pasteur, Cambodia United States Centers for Disease Control and Prevention, National Center for Global Health, Division of Malaria and Parasitic Diseases, United States Foundation for Innovative New Diagnostics, Switzerland WHO, Global Malaria Programme, Switzerland Army Malaria Institute, Australia Hospital for Tropical Diseases, United Kingdom WHO, Global Malaria Programme, Switzerland Research Institute of Tropical Medicine, Philippines Institut Pasteur of Bangui, Central African Republic Université Cheikh Anta Diop, Senegal Universidad Peruana Cayetano Heredia Instituto de Medicina Tropical, Peru Queensland University of Technology, Australia United States Centers for Disease Control and Prevention, National Center for Global Health, Division of Malaria and Parasitic Diseases, United States Foundation for Innovative New Diagnostics, Switzerland Foundation for Innovative New Diagnostics, Switzerland Médecins sans Frontières, Netherlands Department of Medical Research, Myanmar United States Centers for Disease Control and Prevention, National Center for Global Health, Division of Malaria and Parasitic Diseases, United States Research Institute of Tropical Medicine, Philippines Research Institute of Tropical Medicine, Philippines Research Institute of Tropical Medicine, Philippines Queensland Institute of Medical Research, University of Queensland, Australia Institut Pasteur, Madagascar; Institut Pasteur, Cambodia Centro Internacional de Entrenamiento e Investigaciones Médicas, Colombia

VI II

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Sina Nhem Bernhards Ogutu Pamela Onyor Wellington Oyibo Mark Perkins

Institut Pasteur, National Malaria Centre, Cambodia Kenya Medical Research Institute, Kenya Kenya Medical Research Institute, Kenya University of Lagos, Nigeria Foundation for Innovative New Diagnostics, Switzerland

Roxanne Rees-Channer Consultant, Foundation for Innovative New Diagnostics, Hospital for Tropical Diseases, United Kingdom Muth Sinuon Jobel Sornillo Man Somnang Julie Vercruysse Scott Wilson National Malaria Centre, Cambodia Research Institute of Tropical Medicine, Philippines Institut Pasteur, National Malaria Centre, Cambodia Foundation for Innovative New Diagnostics, Switzerland United States Centers for Disease Control and Prevention, National Center for Global Health, Division of Malaria and Parasitic Diseases, United States

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

IX

Abbreviations CDC ELISA FIND HRP2 ISO PCR PDS pLDH Pf Pv Pvom RDT TDR United States Centers for Disease Control and Prevention enzyme-linked immunosorbent assay Foundation for Innovative New Diagnostics histidine-rich protein 2 International Organization for Standardization polymerase chain reaction panel detection score Plasmodium lactate dehydrogenase Plasmodium falciparum Plasmodium vivax Plasmodium vivax, ovale, malariae rapid diagnostic test (for the purposes of this report, immunochromatographic lateral flow devices for the detection of malaria parasite antigens) Special Programme for Research and Training in Tropical Diseases sponsored 
by UNICEF, UNDP, the World Bank and WHO

X

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

1. SUMMARY OF PERFORMANCE OF RAPID DIAGNOSTIC TESTS FOR MALARIA: WHO PRODUCT TESTING ROUNDS 1–5 1.1. Introduction WHO estimates that half the world’s population is at risk of malaria. In 2012, there were an estimated 207 million cases (with an uncertainty range of 135 million to 287 million) and an estimated 627 000 deaths (with an uncertainty range of 473 000 to 789 000). Approximately 90% of all malaria deaths occur in sub-Saharan Africa, and 77% occur in children under 5 years. Malaria remains endemic in 104 countries, and, while parasite-based diagnosis is increasing, most suspected cases of malaria are still not properly confirmed, resulting in over-use of antimalarial drugs and poor disease monitoring (1). WHO recommends that malaria case management be based on parasite diagnosis in all cases (2). The use of antigendetecting rapid diagnostic tests (RDTs) is a vital part of this strategy, forming the basis for extending access to malaria diagnosis by providing parasite-based diagnosis in areas where good-quality microscopy cannot be maintained. The number of RDTs available and the scale of their use have increased rapidly over the past few years; however, limitations of field trials and the heterogeneous nature of malaria transmission have limited the availability of the good-quality data on performance that national malaria programmes require to make informed decisions on procurement and implementation, and it is difficult to extrapolate the results of field trials to different populations and times. Therefore, in 2006, the WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Foundation for Innovative New Diagnostics (FIND) launched a programme to systematically evaluate and compare the performance of commercially available malaria RDTs. The results of WHO’s malaria RDT product testing have been published annually since 2009 and form the basis of the procurement criteria of WHO, other United Nations agencies, the Global Fund to Fight AIDS, Tuberculosis and Malaria, national governments and nongovernmental organizations. The data have guided procurement decisions, which, in turn, have shifted markets towards better-performing tests1 and are driving overall improvements in the quality of manufacturing. This summary presents an overview of the results of rounds 1–5 of malaria RDT product testing and key concepts for understanding and using the results. It is published in conjunction with the release of the full report on round 5. The results of all rounds of testing should be considered as a single data set. The separate, full reports of each round (3–6) should be consulted for further details of methods, product performance and interpretation of the results.

1.2. The WHO product testing programme The RDT evaluations summarized here were performed in collaboration by WHO, TDR, FIND, the United States Centers for Disease Control and Prevention (CDC) and other partners.1 All companies that manufacture according to the ISO 13485:2003 quality system standard were invited to submit one to three products for evaluation in the programme. In each round of testing, products are evaluated against geographically diverse, cryopreserved Plasmodium falciparum and P. vivax clinical samples diluted to 200 and 2000 parasites/µL and with consistently comparable concentration ranges of histidine-rich protein II (HRP2), Plasmodium lactate dehydrogenase (pLDH) and aldolase determined by quantitative enzyme-linked immunosorbent assay (ELISA) (Annex S1). In the first round of testing, 41 products from 21 manufacturers were evaluated against prepared blood panels of cultured P. falciparum parasites, while 29, 50, 48 and 42 products from 13, 23, 27 and 34 manufacturers were evaluated in rounds 2, 3, 4 and 5, respectively. Of these 210 products, 206 progressed to testing against panels of patient-derived P. falciparum and P. vivax parasites and a parasite-negative panel. Thermal stability was assessed after 2 months of storage at elevated temperature and humidity, and a descriptive assessment of ease of use was made. Many manufacturers have decided voluntarily to submit products to one or more rounds of testing, and, in round 5, a requirement was instituted to resubmit products for re-evaluation within 5 years of original testing (Table S1). Of the 206 fully evaluated products, 32 have been evaluated twice, 11 have been evaluated three times and two evaluated four times in rounds 1–5. Of the 147 unique products tested in the programme, 36 detect P. falciparum alone, 101 detect and differentiate P. falciparum from non-P. falciparum malaria (either pan-specific or species-specific for P. vivax or P. vivax, ovale and malariae), 9 detect P. falciparum and non-P. falciparum malaria without distinguishing between them, and one product was designed to detect P. vivax only. Manufacturers submitted two lots of each product for evaluation. When the same products (7 ) were resubmitted in subsequent rounds of testing, the second set of results replaced those from the earlier round. Thus, the performance of some tests in the results below differs from that reported in rounds 1–4. Of the 22 products due for compulsory retesting in round 5, 10 were submitted (Table S1). Round 1 products that were not 1

See full reports of rounds 1–5 (3–6) for lists of collaborating partners.

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Summary Rounds 1-5 1

resubmitted have been removed from the figures and tables in this summary performance document. The aim of the evaluation is to provide comparative data on the performance of the submitted production lots of each product. These data will be used to guide procurement decisions by WHO, other United Nations agencies and national governments and constitute the laboratory evaluation component of the WHO prequalification process for malaria RDTs (8). Product testing is part of a continuing programme of work to improve the quality of RDTs in use and to ensure reliable malaria diagnosis in areas where malaria is prevalent. A sixth round of product testing will begin in June 2014.

1.3.  Panel detection score and other results of the evaluation The results (summarized in Figs S1–S3 and Tables S2 and S3) provide comparative data on two lots of products against a panel of parasite samples diluted to a low parasite density (200 parasites/µL) and a higher parasite density (2000 or 5000 parasites/µL). The former is well below the mean parasite density found in many populations with endemic malaria and is considered close to the threshold that must be detected in order reliably to identify clinical malaria in many settings (9). For the purposes of this report, the main measure of performance is the panel detection score (PDS);1 for each RDT evaluated, the PDS is measured separately at the 1

Termed “detection rate” in the full report of round 1, published in 2009.

Box 1: Example calculation of panel detection score and positivity rate for product A against a sample density of 200 parasites/µL The first reading was at the minimum time specified by the manufacturer; the second reading was up to 30 min latera. A sample is considered detected only if all first test readings, from both lots, are positive, i.e. readings a, b, c and d must be positive.

Product A Lot 1 Lot 2

Test 1

Test 2

Test 3

Test 4

a Reading 1 a second

b Reading 2 Reading 1 Reading 2

c Reading 1 Reading 2

d Reading 1 Reading 2

Detected if 4 positive first readings

reading results are for internal use only

P. falciparum sample 1 2 3

a

b

c

d

+ + +

+

+ +

+ + +

Sample NOT detected Sample NOT detected Sample detected

In this example, only one of three samples was positive all four times it was tested; the PDS is therefore 1/3 = 33%. The positivity rate is calculated as the percentage of all tests of a particular product that returned a positive test result at the manufacturers’ recommended minimum reading time when tested against a P. falciparum or P. vivax sample. In the above example, the positivity rate is: 9/12 = 75%. The positivity rate is always greater than the PDS, except when the PDS and the positivity rate are both 100%.

2

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Box 2: Performance measures in WHO product testing and in field settings: PDS versus clinical sensitivity WHO Malaria RDT Product Testing Primary performance measure: PDS indicates which products are likely to be more sensitive in the field, particularly in populations with low‐density infections. Malaria endemic setting Performance measure: sensitivity is the proportion of the population studied who have malaria for whom the test is positive. - high, moderate, low transmission - immune, non-immune - vulnerable groups

20

20

0

00

pa

pa

ra s

ra s

ite

ite

s/

s/

μL

μL Patients have varying parasite density. Most RDTs for P. falciparum and P. vivax perform well for a parasite density > 2000 parasites/μL, but clinically significant densities < 200 parasites/μL may be missed. The “overall” test performance will nevertheless be classified as very good in a field evaluation.

Reference panels: two fixed parasite densities allows discrimination in RDT performance.

lower and the higher parasite density. The summary figures also show the false-positive rates against blood samples containing no malaria parasites or known markers of other diseases and the rate of invalid results. The PDS is the percentage of malaria samples in the panel that give a positive result in two RDTs per lot at the lower parasite density or by a single RDT per lot at the higher parasite density. As each sample is tested with RDTs from two lots, for a sample to be positive at the lower parasite density, it must show a positive result in four tests (two RDTs per lot for two lots); at the higher parasite density, it must show a positive result in two tests (one RDT per lot for two lots). Thus, the PDS is a combined measure of positivity rate, incorporating inter-test and inter-lot consistency. As all tests performed on each sample must show a positive result for the sample to be considered positive, the PDS for a given RDT will usually be lower than a simple positivity rate per panel, measured by comparing the number of positive tests among all tests performed per panel. The PDS is also different from clinical sensitivity: the ability of the test to detect malaria infection in a given population of infected patients. Boxes 1 and 2 illustrate how the PDS is calculated and how it differs from a simple positivity rate for all samples tested and from clinical sensitivity in a population.

The PDS for a given RDT is different from the clinical sensitivity of that RDT (also called the true positive rate), which is a measure of the proportion of people known to have the disease who test positive for it. The sensitivity of malaria RDTs is highly dependent on local conditions, including the parasite density in the population; it therefore varies among populations with different levels of transmission, as their level of immunity affects the parasite density at which they exhibit symptoms that warrant a diagnostic test. Where transmission rates are low, the parasite densities in people with symptoms of malaria are likely to be low, and tests will be less sensitive. Test performance at 200 parasites/µL is therefore particularly important. The results in this report show the comparative performance of RDTs and indicate which products are likely to be more sensitive in the field, particularly in populations with low-density infections. In general, as countries reduce the prevalence of malaria and even move towards malaria elimination, detection of low parasite densities becomes increasingly important in case management. As the high PDS at 2000 parasites/µL indicates, the sensitivity of many of these products is similar in populations with higher parasite densities and therefore it is not possible to discriminate RDTs with superior performance.

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Summary Rounds 1-5 3

An important caveat to estimating field sensitivity from the PDS provided in this report is that the panels used include only parasites known to express the target antigens. While non-expression of the target antigens has not been recorded for aldolase or pLDH, it is known that parasites that infect people in some areas of South America and India do not express HRP2 (10, 11). In areas where HRP2-deleted parasites exist, tests for HRP2 will have greatly reduced sensitivity or be incapable of detecting P. falciparum. In such populations, only tests for pLDH or aldolase in P. falciparum parasites will be effective for diagnosing falciparum malaria. Heat stability (summarized in Table S3) is vital to maintaining the sensitivity of tests in the field. As a result, for procurement, careful consideration must be given to ensure that the products to be used in areas with high temperatures of transport and storage have demonstrated stability in the product testing programme. Requirements vary among countries; for example, if tests are to be deployed in areas where temperatures rarely rise above 30 °C, less emphasis is needed on stability at high temperatures than on other aspects of quality. Ease-of-use requirements depend on the extent of training and the work environment of the users. Particularly in primary health care settings, the simpler the test, the easier it will be to avoid errors in preparation and interpretation. Detailed results can be found in the report of each evaluation (3 –6) and at http://www.who.int/malaria/publications/ diagnostic_testing/en/.

1.4. Summary of outcomes This laboratory-based evaluation provides a comparative, standardized measure of RDT performance for distinguishing between well and poorly performing tests to serve as a basis for procurement decisions by malaria control programmes and to guide United Nations procurement policy. In round 5, the proportion of tests that achieved a PDS ≥ 75% at 200 parasites/µL is comparable to those in rounds 3 and 4 for P. falciparum (78.6%); that for P. vivax, 42.4%, is similar to that in round 4. Several RDTs in the five rounds of testing consistently detected malaria at a low parasite density (200 parasites/µL), had low false-positive rates, are stable at tropical temperatures, are relatively easy to use and can detect P. falciparum or P. vivax infections or both. Although the performance of the products varied widely at low parasite density (200 parasites/µL), all products had a high rate of detection of P. falciparum at 2000 or 5000 parasites/µL, as did the majority of products for P. vivax at 2000 parasites/µL. P. falciparum tests that target the HRP2 antigen had the highest detection rates, and two previously evaluated tests that target pan-pLDH for detection of Plasmodium spp. infection also achieved a good PDS. In round 5, the two poorest performing tests for detection of P. falciparum were based on P. falciparum -specific pLDH detection. Thus, the choice of well-performing pLDH-based P. falciparum tests remains limited, as it does for pan-only-specific tests. Test performance sometimes varied between lots and widely between similar products, confirming the advisability of testing lots after purchase and before use in the field. Furthermore, anomalies that interfered with test interpretation were regularly recorded during round 5 (Annex S2). All products had issues with red background and with incomplete clearing, and cases of samples failing to flow or migrate on the RDT were reported for 62% of products. Ninety-eight percent of the RDTs evaluated in round 5 were in cassette format. With regard to products retested under the compulsory resubmission requirement, one showed improved (4.8%) detection of P. falciparum and one improved (4.3%) detection of P. vivax, while six and two had diminished performance (> 5% decrease) for detection of P. falciparum (mean, 13.9%; median, 8.4%) and P. vivax (mean, 8.5%), respectively. All products except one had the same or lower false-positive rates (mean improvement, 3.7%).

4

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

1.5.  How can product testing results inform RDT procurement and use? Accurate diagnosis is vital to good malaria case management, whether based on microscopy or RDTs. The results of this report should be used to identify a short list of RDTs for procurement for use in settings where good microscopy is not available or appropriate. Box 3 lists WHO’s minimum criteria for RDT selection, and Annex S3 provides a step-by-step approach to selecting an RDT, taking into consideration local malaria transmission and illness where the tests will be used (e.g. Plasmodium species, target antigen, parasite densities, climate) and other important considerations, including ease of use in the field (Annex S2), training or retraining requirements and lot testing.1 The tabular results in TableS2 are colour-coded to reflect achievement of WHO performance requirements for RDT procurement, and a web-based tool that allows filtering of product testing results by various parameters to assist in selecting products with the performance characteristics most suitable for a country’s health programme is available and maintained by FIND (12). Comprehensive guidance on several aspects of procurement can be found in Good practices for selecting and procuring rapid diagnostic tests for malaria and guidance on implementation in Universal access to malaria diagnosis (13, 14).

1.6.  Product testing and WHO programme for prequalification of diagnostics and medical devices The WHO prequalification of diagnostics and medical devices programme uses the results of product testing as the laboratory evaluation component of the prequalification process for malaria RDTs. These data are used to set priorities for dossier review and inspection. Although prequalification is not currently a requirement for WHO procurement, manufacturers are encouraged to apply for it. A list of prequalified diagnostics, including malaria RDTs, is available at http:// www.who.int/diagnostics_laboratory/evaluations/PQ_list/en/.

1

The WHO-FIND malaria RDT evaluation programme provides lottesting capacity in two regional laboratories free of charge; it can be accessed at malaria_rdt@who.int and info@finddiagnostics.org.

Box 3: WHO selection criteria for the procurement of RDTs Products should be selected in line with the following set of criteria, based on the results of the assessment of the WHO Malaria RDT Product Testing Programme: (A) For the detection of Plasmodium falciparum (Pf) in all transmission settings the panel detection score (PDS) against Pf samples should be at least 75% at 200 parasites/μL. (B) For the detection of Plasmodium vivax (Pv) in all transmission settings the panel detection score (PDS) against Pv samples should be at least 75% at 200 parasites/μL. (C) The false positive rate should be less than 10%. (D) The invalid rate should be less than 5%. Only products meeting performance criteria outlined in A,B,C and D are recommended for procurement

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Summary Rounds 1-5 5

6

Panel detection scorea 100 10 20 30 40 50 60 70 80 90 0 Core™ Malaria Pan Pf MAL-190024 BIOCREDIT Malaria pf(HRP II) HR0100 CareStart™ Malaria HRP2/pLDH Pf test G0181 Falcivax™ Rapid Test for Malaria Pv/Pf 50300025 diagnosticks MALARIA (Pan/Pf) Cassette MPNFWBC1007.4 Core™ Malaria Pv/Pf MAL-190022 Core™ Malaria Pf MAL-190020 SD BIOLINE Malaria Ag Pf/ Pf/ Pv 05FK100 SD BIOLINE Malaria Ag Pf/Pv 05FK80/05FK83 Paracheck® Pf - Rapid test for P. falciparum Malaria Device Ver. 3 302030025 First Response® Malaria Ag P. falciparum (HRP2) Card Test I13FRC SD BIOLINE Malaria Ag Pf 05FK50/05FK53 MD Malaria Pf/Pan(pLDH) test MDMALLDH001 Malaria pf (HRP II) / (PAN-pLDH) Antigen Detection Test Device MFV-124R IND ONE STEP MALARIA ANTIGEN P.f/Pan TEST 535-10 FirstSign™ Malaria Pf 2100CB-25 One Step Malaria P.F Test (cassette) 522352 SD BIOLINE Malaria Ag P.f/Pan 05FK60/05FK63 BIONOTE MALARIA P.f.& Pan Ag Rapid Test Kit RG19-08 diagnosticks MALARIA (Pan/Pv/Pf) Cassette MPNVFC1007.5 Paramax-3 Rapid Test for Malaria Pan/Pv/Pf (device) 50320025 ICT Malaria Dual Test ML03 Core™ Malaria Pan/Pv/Pf MAL-190026 BIONOTE MALARIA P.f.& P.v. Ag Rapid Test Kit RG19-12 NanoSign Malaria Pf/Pan Ag 3.0 RMAP10 Humasis Malaria P.f/P.v Antigen Test AMFV-7025 RAPID 1-2-3® HEMA CASSETTE MALARIA PF/PV TEST MAL-PFV-CAS/25(100) Parascreen® Device - Rapid test for Malaria Pan/Pf 50310025 OnSight™ - ParaQuick-2 (Pv,Pf) Malaria Test 537-25-DB One Step Malaria P.F/P.V Test (Cassette) 523352 CareStart™ Malaria HRP2 (Pf) G0141 diagnosticks- Malaria (Pv/Pf) Cassette KMVFC6002 SD BIOLINE Malaria Ag Pf/ Pan 05FK66 CareStart™ Malaria HRP2/pLDH (Pf/Pv) COMBO G0161 Medisensor Malaria HRP2/pLDH (Pf/Pv) COMBO M161 Humasis Malaria P.f/Pan Antigen Test AMAL-7025 CareStart™ Malaria HRP2/pLDH (Pf/PAN) COMBO G0131 DIAQUICK Malaria P.f/Pan Cassette Z11200CE NG-Test MALARIA Pf/Pan (pLDH) NG-MAL-W23-001 HiSens Malaria Ag P.f/VOM Combo Card HR3323 HiSens Malaria Ag P.f/P.v Combo Card HR3123 Wondfo One Step Malaria P.f Test W37-C CareStart™ Malaria HRP2/pLDH (Pf/VOM) COMBO G0171 Medisensor Malaria HRP2/pLDH (Pf/VOM) COMBO M171 Advantage P.f. Malaria Card IR016025 Malaria Pf/Pan One Step Rapid Test RT 20222 FirstSign™ - ParaView-3 (Pan+Pv+Pf) Malaria Test 2103 CB-25 Clearview® Malaria Dual VB20 CareStart™ Malaria pLDH 3 Line Test G0121 Trusty™ Malaria Antigen P.f. test A03-11-322 Trusty™ Malaria Antigen P.f./p.v. test A03-12-322 diagnosticks- Malaria (Pf) Cassette WB KMFC6001 Advanced™ Quality Rapid Malaria Test (Pf/Pan) ITP11005 SD BIOLINE Malaria Ag P.f. (HRP2/pLDH) - 05FK90 FirstSign™ - ParaView (Pan+Pf) Malaria Test 2101CB-25 HiSens Malaria Ag Pf HRP2 Card HR3023 Wondfo® One Step Malaria P.f/P.v Whole Blood Test W056-C CareStart™ Malaria Screen G0231 Advanced Quality™ One Step Malaria P.f/P.v Tri-Line Test ITP11003 TC40 ICT Diagnostics Malaria P.f. ML01 Vikia® Malaria Ag Pf/Pan 412499 OnSite Pf/Pan Ag Rapid Test R0113C OnSite Pf Ag Rapid Test R0114C BIONOTE MALARIA P.f. Ag Rapid Test Kit RG19-11 ABON™ Plus Malaria P.f/Pan Rapid Test Device (Whole Blood) IMA-T402 First Response® Malaria pLDH/HRP2 Combo Test I16FRC Malaria Pf (HRPII) PV (PLDH) Antigen Detection Test Device GM006 NanoSign Malaria Pf Ag RMAF10 ParaHIT® - f (Device) 55IC102-50 ParaHIT - Total Ver. 1.0 (Device) 55IC204-10 Advantage Malaria Pan + Pf Card IR231025 CareStart™ Malaria pLDH (PAN) G0111

a Panel

b Clean-negative,

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Figure S1: Malaria RDT performance in phase 2 of rounds 2-5 against wild-type (clinical) samples containing P. falciparum at low (200) and high (2000-5000) parasite density (parasites/μL) and clean-negative samples

detection score: A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive. blood samples from healthy volunteers with no known current illness or blood abnormality. * Indicates tests that also detect other non-P. falciparum parasites

OnSite Pf/Pan Ag Rapid Test R0113C OnSite Pf Ag Rapid Test R0114C BIONOTE MALARIA P.f. Ag Rapid Test Kit RG19-11 ABON™ Plus Malaria P.f/Pan Rapid Test Device (Whole Blood) IMA-T402 First Response® Malaria pLDH/HRP2 Combo Test I16FRC Malaria Pf (HRPII) PV (PLDH) Antigen Detection Test Device GM006 NanoSign Malaria Pf Ag RMAF10 ParaHIT® - f (Device) 55IC104-50 ParaHIT - Total Ver. 1.0 (Device) 55IC204-10 Advantage Malaria Pan + Pf Card IR231025 CareStart™ Malaria pLDH (PAN) G0111 Maleriscan® Malaria P.f/PAN (Pv, Pm, Po) 3 Line Antigen Test MAT-PF/PAN-50 GenBody™ Malaria Pf/Pan Ag MALAG100 Vision Malaria Pf VB01 HiSens Malaria Ag Pf/Pv (HRP2/pLDH) Card HR2923 CareStart™ Malaria/Pregnancy Combo (pLDH/HRP2/HCG) G0221 Surestep™ Easy Malaria Pf/Pan Rapid Test Device IMA-T402 Maleriscan ® Malaria P.f Antigen Test MAT-PF-50 EzDx™ Malaria Pan/Pf Rapid Test Detection kit RK MAL 001 Clearview® Malaria Combo VB11 Malascan™ Device - Rapid test for Malaria Pf/Pan 50402025 IMMUNOQUICK CONTACT falciparum 0519K25 Clearview® Malaria pLDH 70884025 ASAN Easy Test® Malaria Pf/Pan Ag AM4650-K ParaHIT® -f (Dipstick) 55IC103-50 diagnosticks- Malaria (Pf) Dipstick WB KMFD6007 Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device MFV-124V OnSite Pf/Pv Ag Rapid Test R0112C KHB® Malaria Ag P.f Rapid Test KH-R-06-20 Advantage Malaria Card IR211025 Advantage Pan Malaria Card IR013025 BioTracer™ Malaria Pf/PAN Rapid Card 17012 BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) C30RHA25 ParaHIT - Total Ver. 1.0 (Dipstick) 55IC203-10 ICT MALARIA COMBO ML02 IMMUNOQUICK CONTACT MALARIA +4 0525K25 OnSight™ - Malaria Pf Test 511-25-DB RightSign™ Malaria P.f/Pan Rapid Test Cassette IMPN-C52 Immunoquick Malaria Falciparum 0502_K25 Paracheck® Pf - Rapid test for P. falciparum Malaria Dipstick (vER.3) 302040025 ABON Malaria Pan/P.f. Rapid Test Device IMA-B402 MeDiPro Malaria Ag HRP2/pLDH Combo IR-0051K Genedia® Malaria P.f/Pan Ag Rapid Test 20-0146-01 ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25 ParaHIT® total (dipstick) 55IC201-10 Malaria pf (HRP II)/PAN (pLDH) Antigen Detection Test Device 1-13-101-1 ParaHIT®fV Rapid test for P. falciparum and P.vivax Malaria - Device 55IC402-50 AZOG Malaria pf (HRPII)/pf (LDH)/ (PAN-LDH) Antigen Detection Device MFV-124F AZOG hCG Malaria Detection Test Device MPT-124 IND ONE STEP MALARIA ANTIGEN P.f 535-11 Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit A03-18-322 Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device 1-13-101-3 Malaria Pan Test MAL-W23N-001 Advanced Quality™ One Step Malaria P.f Test ITP11002TC1/TC40 Maleriscan® Malaria Pf/Pv MAT-50 OptiMAL-IT 710024 Malaria pf (pLDH) / PAN-pLDH Test Device MFV-124 Malaria Pf/Pv GM002 Malaria Pf/ PAN GM004 One Step Malaria P.f/Pan Test W56-C ABON™ Malaria P.f. Rapid Test Device (Whole Blood) IMA-402 First Response® Malaria Ag pLDH I12FRC30 Advantage Mal Card IR221025 FirstSign™ - PanCheck (Pan) Malaria Test 2104 CB-25 OnSight™ - PanScreen (Pan) Malaria Test 539-25-DB HiSens Malaria Ag P.f/P.v Card HR2823 Parabank™ Device - Rapid test for Malaria Pan 50301025 SD BIOLINE Malaria Ag 05FK40 diagnosticks MALARIA (Pan) Cassette MPNWBC1007.3 NanoSign Malaria Pf/Pv Ag RMAD10

0

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200 parasites/µL (HRP2) 200 parasites/µL (non-HRP2) 2000 parasites/µL (HRP2) 2000 parasites/µL (non-HRP2) False-positive Plasmodium spp. rate (%) Invalid rate (%)

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

False-positive Plasmodium spp. rate on clean-negative samplesb (%) and invalid rate (%)

7

Summary Rounds 1-5

8

a Panel

Panel detection scorea 100 10 20 30 40 50 60 70 80 90 0

b Clean-negative

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Figure S2: Malaria RDT performance in phase 2 of rounds 2-5 against wild-type (clinical) samples containing P. vivax at low (200) and high (2000 or 5000) parasite density (parasites/μL) and clean-negative samples

detection score - A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive. - blood samples from healthy volunteers with no known current illness or blood abnormality.

Advantage Malaria Pan + Pf Card IR231025 Advantage Pan Malaria Card IR013025 Humasis Malaria P.f/P.v Antigen Test AMFV-7025 OnSite Pf/Pv Ag Rapid Test R0112C One Step Malaria P.F/P.V Test (Cassette) 523352 SD BIOLINE Malaria Ag 05FK40 NanoSign Malaria Pf/Pan Ag 3.0 RMAP10 OptiMAL-IT 710024 SD BIOLINE Malaria Ag Pf/ Pf/ Pv 05FK100 BIONOTE MALARIA P.f.& P.v. Ag Rapid Test Kit RG19-12 SD BIOLINE Malaria Ag Pf/Pv 05FK80/05FK83 CareStart™ Malaria/Pregnancy Combo (pLDH/HRP2/HCG) G0221 CareStart™ Malaria HRP2/pLDH (Pf/PAN) COMBO G0131 Advantage Mal Card IR221025 CareStart™ Malaria HRP2/pLDH (Pf/Pv) COMBO G0161 Medisensor Malaria HRP2/pLDH (Pf/Pv) COMBO M161 SD BIOLINE Malaria Ag Pf/ Pan 05FK66 First Response® Malaria Ag pLDH I12FRC30 SD BIOLINE Malaria Ag Pv 05FK70 SD BIOLINE Malaria Ag P.f/Pan 05FK60/05FK63 Humasis Malaria P.f/Pan Antigen Test AMAL-7025 Malaria Pf/Pan One Step Rapid Test RT 20222 CareStart™ Malaria pLDH 3 Line Test G0121 CareStart™ Malaria HRP2/pLDH (Pf/VOM) COMBO G0171 Medisensor Malaria HRP2/pLDH (Pf/VOM) COMBO M171 CareStart™ Malaria pLDH (PAN) G0111 OnSite Pf/Pan Ag Rapid Test R0113C CareStart™ Malaria Screen G0231 BIONOTE MALARIA P.f.& Pan Ag Rapid Test Kit RG19-08 Falcivax™ Rapid Test for Malaria Pv/Pf 50300025 One Step Malaria P.f/Pan Test W56-C Clearview® Malaria pLDH 70884025 DIAQUICK Malaria P.f/Pan Cassette Z11200CE FirstSign™ - PanCheck (Pan) Malaria Test 2104 CB-25 ParaHIT - Total Ver. 1.0 (Device) 55IC204-10 EzDx™ Malaria Pan/Pf Rapid Test Detection kit RK MAL 001 RAPID 1-2-3® HEMA CASSETTE MALARIA PF/PV TEST MAL-PFV-CAS/25(100) HiSens Malaria Ag P.f/P.v Combo Card HR3123 OnSight™ - PanScreen (Pan) Malaria Test 539-25-DB BioTracer™ Malaria Pf/PAN Rapid Card 17012 BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) C30RHA25 HiSens Malaria Ag P.f/VOM Combo Card HR3323 HiSens Malaria Ag Pf/Pv (HRP2/pLDH) Card HR2923 First Response® Malaria pLDH/HRP2 Combo Test I16FRC Malaria Pf (HRPII) PV (PLDH) Antigen Detection Test Device GM006 NG-Test MALARIA Pf/Pan (pLDH) NG-MAL-W23-001 Maleriscan® Malaria P.f/PAN (Pv, Pm, Po) 3 Line Antigen Test MAT-PF/PAN-50 Parabank™ Device - Rapid test for Malaria Pan 50301025 ParaHIT - Total Ver. 1.0 (Dipstick) 55IC203-10 IND ONE STEP MALARIA ANTIGEN P.f/Pan TEST 535-10 FirstSign™ - ParaView (Pan+Pf) Malaria Test 2101CB-25 Core™ Malaria Pv/Pf MAL-190022 Advanced™ Quality Rapid Malaria Test (Pf/Pan) ITP11005 Clearview® Malaria Dual VB20 Malascan™ Device - Rapid test for Malaria Pf/Pan 50402025 GenBody™ Malaria Pf/Pan Ag MALAG100 diagnosticks MALARIA (Pan) Cassette MPNWBC1007.3 diagnosticks MALARIA (Pan/Pf) Cassette MPNFWBC1007.4

FirstSign™ - ParaView (Pan+Pf) Malaria Test 2101CB-25 Core™ Malaria Pv/Pf MAL-190022 Advanced™ Quality Rapid Malaria Test (Pf/Pan) ITP11005 Clearview® Malaria Dual VB20 Malascan™ Device - Rapid test for Malaria Pf/Pan 50402025 GenBody™ Malaria Pf/Pan Ag MALAG100 diagnosticks MALARIA (Pan) Cassette MPNWBC1007.3 diagnosticks MALARIA (Pan/Pf) Cassette MPNFWBC1007.4 Paramax-3 Rapid Test for Malaria Pan/Pv/Pf (device) 50320025 diagnosticks- Malaria (Pv/Pf) Cassette KMVFC6002 FirstSign™ - ParaView-3 (Pan+Pv+Pf) Malaria Test 2103 CB-25 ICT Malaria Dual Test ML03 RightSign™ Malaria P.f/Pan Rapid Test Cassette IMPN-C52 Trusty™ Malaria Antigen P.f./p.v. test A03-12-322 OnSight™ - ParaQuick-2 (Pv,Pf) Malaria Test 537-25-DB Parascreen® Device - Rapid test for Malaria Pan/Pf 50310025 ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25 ParaHIT®fV Rapid test for P. falciparum and P.vivax Malaria - Device 55IC402-50 ASAN Easy Test® Malaria Pf/Pan Ag AM4650-K Advantage Malaria Card IR211025 Wondfo® One Step Malaria P.f/P.v Whole Blood Test W056-C MD Malaria Pf/Pan(pLDH) test MDMALLDH001 Core™ Malaria Pan Pf MAL-190024 IMMUNOQUICK CONTACT MALARIA +4 0525K25 Genedia® Malaria P.f/Pan Ag Rapid Test 20-0146-01 HiSens Malaria Ag P.f/P.v Card HR2823 diagnosticks MALARIA (Pan/Pv/Pf) Cassette MPNVFC1007.5 Core™ Malaria Pan/Pv/Pf MAL-190026 ParaHIT® total (dipstick) 55IC201-10 NanoSign Malaria Pf/Pv Ag RMAD10 Malaria pf (pLDH) / PAN-pLDH Test Device MFV-124 ABON™ Plus Malaria P.f/Pan Rapid Test Device (Whole Blood) IMA-T402 ICT MALARIA COMBO ML02 Vikia® Malaria Ag Pf/Pan 412499 Clearview® Malaria Combo VB11 Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit A03-18-322 Malaria Pf/ PAN GM004 Malaria pf (HRP II)/PAN (pLDH) Antigen Detection Test Device 1-13-101-1 MeDiPro Malaria Ag HRP2/pLDH Combo IR-0051K Surestep™ Easy Malaria Pf/Pan Rapid Test Device IMA-T402 Malaria pf (HRP II) / (PAN-pLDH) Antigen Detection Test Device MFV-124R AZOG Malaria pf (HRPII)/pf (LDH)/ (PAN-LDH) Antigen Detection Device MFV-124F ABON Malaria Pan/P.f. Rapid Test Device IMA-B402 Maleriscan® Malaria Pf/Pv MAT-50 AZOG hCG Malaria Detection Test Device MPT-124 Malaria Pan Test MAL-W23N-001 Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device 1-13-101-3 Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device MFV-124V Malaria Pf/Pv GM002 Advanced Quality™ One Step Malaria P.f/P.v Tri-Line Test ITP11003 TC40

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200 parasites/µL 2000 parasites/µL False-positive Plasmodium spp. rate (%) Invalid rate (%)

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

False-positive Plasmodium spp. rate on clean-negative samplesb (%) and invalid rate (%)

9

Summary Rounds 1-5

Figure S3: Panel detection score of malaria combination RDTs, meeting WHO procurement criteria for false-positive and invalid rates, in phase 2 of rounds 2-5 against wild-type (clinical) samples containing P. falciparum and P. vivax at low (200) parasite density (parasites/μL)

Panel detection score P. vivaxa

100 80 60 40 20 0

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Panel detection score P. falciparum

a Panel

detection score - A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive.

10

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Table S1: Product resubmissions: WHO malaria RDT product testing rounds 1—5 Product re-submission Manufacturer Product name CareStart™ Malaria HRP2/PLDH (Pf/Pv) COMBO CareStart™ Malaria HRP2/PLDH (Pf/VOM) COMBO CareStart™Malaria HRP2 (Pf) CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo CareStart™Malaria pLDH (PAN) EzDx™ Malaria Pan/Pf Rapid Test Detection Kit IMMUNOQUICK® MALARIA falciparum Malaria pf (HRP II) / (PAN-LDH) Antigen Detection Test Devicea Malaria pf (pLDH) / PAN-pLDH Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test NanoSign Malaria Pf/Pan Ag One Step Malaria Pf Test (cassette) One Step Malaria P.F/P.V Test (Cassette) Onsite Pf Ag Rapid Test Onsite Malaria Pf/Pan Malaria Ag Rapid Test Onsite Malaria Pf/Pv Ag Rapid Test OptiMAL-IT Wondfo One Step Malaria Pf/Pan Whole Blood Test One Step Malaria P.f Testb ICT Malaria Combo Cassette Test ICT Malaria Pf Cassette Test ICT Malaria Dual Test Advanced Quality™ One Step Malaria Pf Test Humasis Malaria Pf/Pan Antigen Test Advantage Pan Malaria Card Advantage Mal Card Advantage P.f Malaria Card Paracheck® Pf Device - Rapid test for P. falciparum Malaria (Ver. 3)c Paracheck® Pf Dipstick - Rapid test for P. falciparum Malaria (Ver.3)c First Response® Malaria Ag Combo (pLDH/HRP2)d First Response Malaria Ag P. falciparum (HRP2) Card Test diagnosticks- Malaria (Pf)Cassette WB SD BIOLINE Malaria Ag SD BIOLINE Malaria Ag Pf/Pan SD BIOLINE Malaria Antigen FirstSign™ - ParaView (Pan+Pf) Malaria Test Malaria Rapid Combo/Clearview® Malaria Combo Malaria Rapid Pf /Clearview ®Malaria Pf Malaria Rapid Dual/Clearview® Malaria Dual Test Device Malascan™ Device - Rapid test for Malaria Pf/Pan Parabank™ Device - Rapid test for Malaria Pan Parascreen™ Device -Rapid test for Malaria Pan/Pf Falcivax Rapid Test for Malaria Pv/Pf (device)

Catalogue No. G0161 G0171 G0141 G0131 G0111 RK MAL 001 0502_K25 MFV-124R MFV-124 MAT-PF/PAN-50 RMAP10 522352 523352 R0114C R0113C R0112C 710024 W56-C W37-C ML02 ML01 ML03 ITP11002TC1/TC40 AMAL-7025 IR013025 IR221025 IR016025 30301025 30302025 I16FRC I13FRC KMFC6001 05FK40 05FK60/05FK63 05FK50/05FK53 2101 CB-25 VB11e VB01 VB20e 50402025 50301025 50310025 50300025 2, 4 2, 4 1 1 1 4, 5 1 1, 3 3, 5 4, 5 3, 4 2, 3, 4 4, 5 2, 3 2, 3, 4, 5 2, 3, 4 1, 3 1, 3 2, 3, 4 1, 3, 4 1, 3 3, 5 1, 3 4, 5 1 1 1 1, 3, 4 1, 3, 4 1, 2, 5 1 2, 5 1, 3 1, 3, 5 1 2, 4 1, 3 1, 3, 5 1, 3, 5 1, 3 1, 3 1, 3, 4, 5 2, 4

Round Voluntary Compulsory

Access BIo, Inc.

5 5 5

Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Biosynex AZOG Bhat Bio-Tech India (P) Ltd. Bioland Blue Cross Bio-Medical (Beijing) Co., Ltd. CTK Biotech, Inc. DiaMed - A Division of Bio-Rad Guangzhou Wondfo Biotech Co. Ltd. ICT INTERNATIONAL InTec Products, Inc. Humasis Co., Ltd.

5

5 5 5 5

J.Mitra & Co. Pvt. Ltd. Orchid Biomedical Systems Premier Medical Corporation Ltd. SSA Diagnostics & Biotech Systems Standard Diagnostics Inc. Unimed International Inc. Vision Biotech (Pty) Ltd / Orgenics (Alere Healthcare (Pty) Ltd subsidaries)

5

5

Zephyr Biomedical Systems a

Round 1 product name error: published - Malaria Pf (HRPII)/pv-LDH) Antigen Detection Test Device Code; corrected product name: Malaria Pf (HRPII/PAN-LDH) Antigen Detection Test Device Code. No change in product code. b In round 2, product did not pass phase-1, therefore results do not feature in summary tables. c Ver.3 was introduced after round 1 d Error in WHO malaria RDT product testing: round 1 report: product code (II6FRC30) should have been ( I16FRC ), as in round 2 e New company acquisition (Alere™), therefore change in product branding and catalogue numbers; VB011 to VB11 and VB020 to VB20. Manufacturer confirmed compliance with product definition.

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Summary Rounds 1-5 11

Pf samplesc

Pv samplesd

Pf samplesc

Pv samplesd

12

Table S2: Malaria RDT phase-2 performance in rounds 2–5 against wild-type (clinical) samples containing P. falciparum (Pf) and P. vivax (Pv) at low (200) and high (2000-5000) parasite density (parasites/μL) and clean-negative samples Panel detection scorea False-positive rates (%) 200 parasites/μL Pf samples Pv samples Pf samples Pv samples Falsepositive non-Pf infectione NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 69.7 85.7 66.0 88.0 30.0 0.0 5.9 37.1 60.0 94.3 99.0 100.0 92.0 100.0 94.0 62.9 97.1 97.1 97.1 97.1 0.0 0.0 0.3 0.3 (389) 1.5 11.4 1.5 0.0 0.0 8.1 0.7 0.0 0.0 0.0 0.0 0.0 0.0 4.4 (135) 2.1 0.0 0.0 0.0 0.0 (139) 6.7 (134) 0.7 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 0.0 (199) 0.0 (197) 0.5 0.7 97.1 0.0 0.0 0.6 0.0 0.0 2.1 2.5 0.7 0.7 0.0 0.0 3.6 (139) 3.6 2.2 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 3.6 NA 5.7 0.0 95.59 1.4 0.0 1.3 0.0 0.0 1.4 3.8 0.0 1.47 0.0 0.0 1.4 4.3 14.71 12.9 0.0 0.0 1.47 2.5 0.0 0.0 0.0 0.0 1.4 0.0 2.9 2.94 1.4 0.0 0.0 0.0 0.0 1.4 0.0 0.0 NA 0.0 0.4 7.7 (233) 0.0 99.1 (231) 2.0 0.9 3.0 0.0 1.0 (198) 0.9 (235) 2.0 0.4 2.2 (231) 1.0 0.0 4.0 (199) 5.1 (234) 6.0 10.6 (235) 0.4 0.0 1.3 11.0 3.5 1.3 0.9 0.0 2.5 2.0 0.0 5.2 (230) 5.1 (235) 0.4 (231) 0.0 0.4 7.3 (234) 8.7 (231) 0.4

Total false-positive ratesb (%) Clean-negative samples False-positive Plasmodium spp. Infectioni 0.0 0.4 0.0 0.5 0.1 0.0 0.0 0.0 0.3 0.3 0.0 0.0 0.2 0.1 0.0 0.3 0.2 0.1 0.7 0.2 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7 0.1 0.2 0.0 0.0 0.4 2.1 0.0 4 5 5 4 3 5 2 3 3 5 2 5 4 2 3 3 5 4 5 4 3 4 2 3 4 4 3 3 3 5 4 5 4 3 4 5 5 5

200 parasites/μL Manufacturer Falsepositive Pf infectionf Falsepositive non-Pf infectiong Falsepositive Pf infectionh

2000 or 5000 parasites/μL

2000 or 5000 parasites/μL

Product

Catalogue number

Invalid rate (%)l

Round

Pf only ABON™ Malaria P.f. Rapid Test Device (Whole Blood) ABON Biopharm (Hangzhou) Co. Ltd InTec Products, Inc. 89.0 99.0 85.9 91.0 98.0 83.8 97.0 88.0 80.0 95.0 94.9 87.0 86.9 81.8 72.0 61.2 79.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 83.7 84.9 94.9 74.0 85.9 95.9 70.4 84.9 80.8 87.9 95.0 88.8 84.0 89.8 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 98.0 100.0 99.0 99.0 100.0 98.0 99.0 100.0 99.0 100.0 99.0 100.0 100.0 99.0 NA 91.8(98) NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 99.0 100.0 99.0 100.0 100.0 100.0 100.0 100.0 99.0 100.0 100.0 100.0 98.0 100.0 93.0 99.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 53.0 NA 93.0 NA 32.7 NA 99.0 NA

Advanced Quality™ One Step Malaria Pf Testj

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

J. Mitra & Co. Pvt. Ltd. RapiGen Inc. Bionote,Inc. Access Bio, Inc. Access Bio, Inc. Vision Biotech (Pty) Ltd Core Diagnostics SSA Diagnostics & Biotech Systems SSA Diagnostics & Biotech Systems Premier Medical Corporation Ltd. Unimed International Inc. HBI Co., Ltd. ICT INTERNATIONAL Biosynex Biosynex IND Diagnostics Inc. Shanghai Kehua Bio-engineering KHB® Malaria Ag P.f Rapid Test KH-R-06-20 Co.,Ltd. Maleriscan ® Malaria P.f Antigen Test MAT-PF-50 Bhat Bio-Tech India (Pte.) Ltd. NanoSign Malaria Pf Ag RMAF10 Bioland, Ltd 522352 Blue Cross Bio-Medical (Beijing) Co., Ltd. One Step Malaria P.F Test (Cassette)j OnSight™ - Malaria Pf Test 511-25-DB Amgenix International, Inc. OnSite Pf Ag Rapid Testj R0114C CTK Biotech, Inc. Paracheck® Pf-Rapid Test for P. falciparum Malaria Device (Ver.3)j 302030025 Orchid Biomedical Systems Paracheck® Pf-Rapid Test for P. falciparum Malaria Dipstick (Ver.3)j 302040025 Orchid Biomedical Systems ParaHIT® - f (Device) 55IC104-50 Span Diagnostics Ltd. ParaHIT® -f (Dipstick) 55IC103-50 Span Diagnostics Ltd. SD BIOLINE Malaria Ag P.f. (HRP2/pLDH)k 05FK90 Standard Diagnostics Inc. SD BIOLINE Malaria Ag Pfj 05FK50/05FK53 Standard Diagnostics, Inc. Trusty™ Malaria Antigen P.f. test A03-01-322 Artron Laboratories Inc. Vision Malaria Pfj VB01 Vision Biotech (Pty) Ltd Wondfo One Step Malaria P.f Testj W 37-C Guangzhou Wondfo Biotech Co. Ltd. Pf and pan ABON Malaria Pan/P.f. Rapid Test Device IMA-B402 ABON Biopharm (Hangzhou) Co. Ltd. ABON™ Plus Malaria P.f/Pan Rapid Test Device (Whole Blood) IMA-T402 ABON Biopharm (Hangzhou) Co. Ltd ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25 LAB-CARE Diagnostics (India) PVT. LTD. Advanced Quality™ Rapid Malaria Test (Pf/Pan) ITP11005 InTec Products, Inc. Advantage Mal Cardj IR221025 J. Mitra & Co. Pvt. Ltd.

Advantage P.f. Malaria Cardj BIOCREDIT Malaria pf(HRP II) BIONOTE MALARIA P.f. Ag Rapid Test Kit CareStart™ Malaria HRP2 (Pf)j CareStart™ Malaria HRP2/pLDH Pf test Clearview® Malaria P.f.j Core™ Malaria Pf diagnosticks- Malaria (Pf) Cassette WB diagnosticks- Malaria (Pf) Dipstick WB First Response® Malaria Ag P. falciparum (HRP2) Card Testj FirstSign™ Malaria Pf HiSens Malaria Ag Pf HRP2 Card ICT Diagnostics Malaria P.f.j IMMUNOQUICK CONTACT falciparum IMMUNOQUICK® MALARIA falciparumj IND ONE STEP MALARIA ANTIGEN P.f

IMA-402 ITP11002TC1/ TC40 IR016025 HR0100 RG19-11 G0141 G0181 VB01 MAL-190020 KMFC6001 KMFD6007 I13FRC 2100CB-25 HR3023 ML01 0519K25 0502_K25 535-11

Table S2 (continued) Panel detection scorea False-positive rates (%) 200 parasites/μL Pf samples Pv samples Pf samples Pv samples Falsepositive non-Pf infectione False-positive Plasmodium spp. Infectioni 0.0 1.7 (231) 4.7 3.0 (199) 0.4 0.0 0.4 0.5 2.5 (199) 3.5 12.7 32.2 (230) 0.0 0.0 0.0 0.0 1.5 0.0 13.6 0.0 0.0 0.0 (138) 4.3 0.7 5.1 (138) 2.2 15.7 4.3 7.9 0.0 (135) 47.9 0.7 0.7 (138) 0.7 (136) 38.6 0.0 0.0 0.0 (139) 90.0 65.7 100.0 94.3 0.5 (399) 9.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (199) 0.5 0.0 (195) 0.0 0.0 0.0 0.0 (199) 8.1 0.0 1.5 0.0 0.0 (199) 1.0 (194) 0.0 0.0 0.0 0.0 0.0 0.0 (69) 1.5 (67) 0.0 0.0 0.0 0.0 7.1 0.0 0.0 1.4 2.9 1.5 0.0 5.9 17.1 0.0 0.0 0.0 35.7 0.0 0.0 (69) 0.0 (68) 40.0 1.5 0.0 0.0 4.3 2.5 2.1 1.3 (235) 0.0 2.6 0.0 (235) 10.6 0.0 0.5 0.9 (235) 3.0 0.4 2.0 1.3 44.0 0.9 5.5 (199) 0.0 81.3 (235) 0.0 0.4 (232) 1.0 (195) 39.4 0.9 0.4 0.0 15.3 0.2 0.3 0.2 0.1 0.0 0.2 0.0 0.0 0.1 0.0 0.0 0.3 0.3 0.2 0.3 0.0 0.0 0.2 0.1 0.0 0.0 0.7 0.0 0.1 0.3 0.0 0.0 0.2 0.3 0.1 0.1 0.0 1.0 1.9 0.0 0.1 0.0 0.1 0.1 5 4 5 3 5 3 5 3 3 3 5 4 3 5 5 5 4 5 5 2 2 5 5 4 3 4 3 5 3 4 5 4 5 3 5 4 4 3 5 3.5 0.0 (390) 0.8 0.0 0.5 2.3 1.5 1.3 1.8 0.0 0.3 0.0 0.0 (394) 0.3 0.3 0.3 0.3 0.0 0.0 0.0 0.0 0.5 (396) 0.3 0.5 1.8 (395) 0.0 2.8 0.0 (3.98) 0.0 (395) 0.0 22.5 0.3 0.0 (398) 1.0 (392) 0.0 0.0 (391) 0.8 0.5 2.9 0.7 (139) 0.0 0.7 0.0 0.7 1.4 (139) 0.7 0.7 2.1 5.7 12.1 33.8 0.5 (198) 0.0 2.0 0.0 (194) 0.0 6.1 0.0 0.5 0.5 0.0 0.0 0.0 0.0 1.4 0.0 0.0 0.0 5.7 7.1 42.7 5.2 0.0 0.0 0.0 0.0 0.0 (69)

Total false-positive ratesb (%) Clean-negative samples

200 parasites/μL Manufacturer Pf samplesc Pv samplesd Pf samplesc Pv samplesd Falsepositive Pf infectionf Falsepositive non-Pf infectiong Falsepositive Pf infectionh

2000 or 5000 parasites/μL

2000 or 5000 parasites/μL

Product

Catalogue number

Invalid rate (%)l

Round

IR231025 AZOG, INC. RapiGEN INC. Bionote,Inc. Bio Focus Co., Ltd. Access Bio, Inc. Access Bio, Inc. Access Bio, Inc. Access Bio, Inc. Vision Biotech (Pty) Ltd Orgenics Ltd.(IS) Core Diagnostics Ltd. SSA Diagnostics & Biotech Systems 90.0 83.0 85.0 87.8 84.0 67.0 20.0 84.0 90.0 93.0 76.5 75.8 94.9 54.6 61.0 95.0 63.3 41.0 39.8 89.0 82.8 95.0 69.4 92.9 6.1 74.3 61.8 54.3 17.1 15.0 75.0 91.4 40.0 5.9 17.1 61.8 0.0 2.9 0.0 2.9 8.6 2.9 91.4 57.1 28.6 2.9 97.1 8.6 100.0 100.0 100.0 96.0 94.0 99.0 100.0 98.0 99.0 98.0 100.0 97.0 95.0 100.0 100.0 97.0 94.9 100.0 97.0 100.0 99.0 100.0 89.9 100.0 100.0 97.1 88.6 100.0 100.0 97.1 94.3 88.2 94.3 85.3 48.6 97.1 94.3 85.3 45.7 97.1 100.0 100.0 40.0 0.0 100.0 100.0 80.0 100.0 100.0 82.9 100.0 97.1 98.0 51.4 100.0 97.1 77.0 93.9 77.0 83.8 90.0 88.9 86.9 82.8 89.0 99.0 77.1 88.6 77.1 94.3 94.3 91.4 88.6 5.7 60.0 26.5 99.0 99.0 94.0 100.0 100.0 100.0 100.0 100.0 99.0 100.0 97.1 100.0 100.0 97.1 100.0 100.0 100.0 91.4 85.7 29.4 62.2 0.0 98.0 88.2

J. Mitra & Co. Pvt. Ltd.

84.0

100.0

100.0

100.0

Advantage Malaria Pan + Pf Card AZOG Malaria pf (HRPII)/pf (LDH)/ (PAN-LDH) Antigen Detection Devicek BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BIONOTE MALARIA P.f.& Pan Ag Rapid Test Kit BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria/Pregnancy Combo (pLDH/HRP2/HCG) CareStart™ Malaria HRP2/pLDH (Pf/PAN) COMBOj CareStart™ Malaria pLDH 3 Line Test CareStart™ Malaria Screen Clearview® Malaria Comboj Clearview® Malaria Dualj Core™ Malaria Pan Pf

MFV-124F

diagnosticks MALARIA (Pan/Pf) Cassette

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

DIALAB GmbH Advy Chemical (Affiliate of Bharat EzDx™ Malaria Pan/Pf Rapid Test Detection kitj RK MAL 001 Serums & Vaccines Ltd. ) First Response® Malaria Ag. pLDH/HRP2 Combo Card Testj I16FRC Premier Medical Corporation Ltd. FirstSign™ ParaView (Pan+Pf)j 2101CB-25 Unimed International Inc. GenBody™Malaria Pf/Pan Ag MALAG100 GenBody Inc. Genedia® Malaria P.f/Pan Ag Rapid Test 20-0146-01 Green Cross Medical Science Corp. (Korea) HiSens Malaria Ag P.f/P.v Card HR2823 HBI Co., Ltd. HiSens Malaria Ag Pf/Pv (HRP2/pLDH) Card HR2923 HBI Co., Ltd. Humasis Malaria P.f/Pan Antigen Testj AMAL-7025 Humasis, Co., Ltd. ICT Malaria Dual Test j ML03 ICT INTERNATIONAL ICT MALARIA COMBOj ML02 ICT INTERNATIONAL IMMUNOQUICK CONTACT MALARIA +4 0525K25 Biosynex IND ONE STEP MALARIA ANTIGEN P.f/Pan TEST 535-10 IND Diagnostics Inc. Malaria Pan Test MAL-W23N-001 Dima • Gesellschaft für Diagnostika mbH Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit A03-18-322 Artron Laboratories Inc. Malaria pf (HRP II) / (PAN-pLDH) Antigen Detection Test Devicej MFV-124R AZOG, Inc. Malaria pf (HRP II)/PAN (pLDH) Antigen Detection Test Device 1-13-101-1 United Biotech, Inc. Malaria pf (pLDH) / PAN-pLDH Test Device j MFV-124 AZOG, Inc. Malaria Pf/ PAN GM004 Genomix Molecular Diagnostics Pvt.Ltd. Malaria Pf/Pan One Step Rapid Test RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. Malascan™ Device - Rapid test for Malaria Pf/Panj 50402025 Zephyr Biomedical Systems MD Malaria Pf/Pan(pLDH) test MDMALLDH001 Medical Diagnostech (Pty) Ltd MeDiPro Malaria Ag HRP2/pLDH Combo IR-0051K Formosa Biomedical Technology Corp. NanoSign Malaria pf/pan Ag 3.0j RMAP10 Bioland Ltd. NanoSign Malaria Pf/Pv Ag RMAD10 Bioland, Ltd NGSARL NG Biotech, Z.A. NG-Test MALARIA Pf/Pan (pLDH) MAL-W23-001

DIAQUICK Malaria P.f/Pan Cassette

C30RHA25 RG19-08 17012 G0221 G0131 G0121 G0231 VB11 VB20 MAL-190024 MPNFWBC 1007.4 Z11200CE

(continued)

Summary Rounds 1-5

13

Pf samplesc

Pv samplesd

Pf samplesc

Pv samplesd

14

Table S2: Malaria RDT phase-2 performance in rounds 2–5 against wild-type (clinical) samples containing P. falciparum (Pf) and P. vivax (Pv) at low (200) and high (2000-5000) parasite density (parasites/μL) and clean-negative samples (continued) Panel detection scorea False-positive rates (%) 200 parasites/μL Pf samples Pv samples Pf samples Pv samples Falsepositive non-Pf infectione 8.4 (383) 0.0 (399) 1.5 0.3 0.8 0.0 0.5 2.0 0.8 1.0 (385) 0.8 0.0 0.0 15.7 (395) 0.5 16.5 0.3 0.3 0.3 0.3 0.3 (399) 0.8 0.3 (391) 0.0 0.5 0.5 0.0 1.5 (391) 0.8 27.3 (399) 1.8 (399) 0.3 0.3 21.5 0.5 1.5 2.0 (399) 92.9 79.4 100.0 100.0 0.0 5.7 0.7 0.0 0.7 0.0 0.7 0.0 0.6 2.9 0.0 0.0 0.7 0.0 (135) 1.4 6.5 (138) 0.7 5.8 (139) 2.5 0.0 0.7 53.6 1.9 0.0 5.7 0.7 8.1 (197) 0.0 85.5 1.5 (197) 1.0 0.5 0.0 0.0 0.0 0.5 0.5 0.5 3.1 (195) 0.0 3.6 (195) 0.5 87.4 (199) 32.5 1.0 0.5 9.0 0.0 2.0 0.5 0.0 4.3 0.0 0.0 0.0 1.5 2.9 0.0 0.0 2.9 0.0 0.0 1.5 1.5 0.0 2.9 0.0 4.3 (69) 2.5 (79) 1.5 2.9 34.3 0.0 0.0 2.9 1.5 0.0 (137) 0.0 0.0 0.0 0.0 0.0 0.7 2.9 0.7 0.0 (130) 0.0 0.0 0.7 (139) 0.0 (194) 0.0 0.5 0.5 0.0 0.0 0.0 (199) 0.5 0.5 0.0 (195) 0.0 0.0 0.5 (199) 0.0 (68) 1.4 20.3 (69) 3.0 (67) 1.5 0.0 1.4 5.7 1.4 0.0 (67) 0.0 0.0 0.0 (69)

Total false-positive ratesb (%) Clean-negative samples False-positive Plasmodium spp. Infectioni 4.1 (195) 1.3 2.0 (198) 0.0 0.0 7.0 4.2 14.0 0.0 1.3 (226) 0.0 1.0 1.3 (235) 18.5 0.0 0.4 (235) 4.0 0.0 0.0 4.0 2.0 7.3 0.4 0.0 1.3 0.0 (230) 0.0 (199) 0.9 (232) 0.9 3.0 (232) 1.5 (199) 0.0 0.0 77.1 3.5 1.3 6.4 4.3 2.4 0.1 0.5 0.1 0.0 0.0 0.1 0.0 0.0 2.8 0.0 0.0 0.3 0.2 0.0 0.2 0.0 0.0 0.0 0.1 0.1 0.0 0.1 0.0 0.0 0.5 0.1 2.5 0.0 0.7 0.4 0.0 0.0 0.0 0.1 0.0 0.1 0.0 3 5 3 4 4 2 5 5 5 4 3 3 5 3 3 5 3 4 4 3 2 4 4 4 4 4 3 5 4 5 2 4 4 5 2 4 5 4

200 parasites/μL Manufacturer Falsepositive Pf infectionf Falsepositive non-Pf infectiong Falsepositive Pf infectionh

2000 or 5000 parasites/μL

2000 or 5000 parasites/μL

Product

Catalogue number

Invalid rate (%)l

Round

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013) Guangzhou Wondfo Biotech Co. Ltd. CTK Biotech, Inc. Diamed - A Division of Bio-Rad Span Diagnostics Ltd. Span Diagnostics Ltd. Span Diagnostics Ltd Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. Standard Diagnostics Inc. Standard Diagnostics Inc. ACON Biotech (Hangzhou) Co. Ltd. IMACCESS S.A.S InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. ASAN Pharmaceutical Co., Ltd Bionote,Inc. Access Bio, Inc. Access Bio, Inc. Core Diagnostics SSA Diagnostics & Biotech Systems Zephyr Biomedicals HBI Co., Ltd. HBI Co., Ltd. Humasis, Co., Ltd. United Biotech, Inc. AZOG, Inc. Genomix Molecular Diagnostics Pvt. Ltd. Genomix Molecular Diagnostics Pvt.Ltd. Bhat Bio-Tech India (P) Ltd. Bhat Bio-Tech India (P) Ltd Medisensor, Inc. Medisensor, Inc. Blue Cross Bio-Medical (Beijing) Co., Ltd. Amgenix International, Inc. CTK Biotech, Inc. Span Diagnostics Ltd. Hema Diagnostic Systems, LLC 86.9 77.8 81.0 92.9 90.8 89.8 98.0 91.0 98.0 89.8 89.8 92.9 60.2 79.8 85.0 40.8 84.0 52.0 90.8 89.8 92.0 92.0 79.6 63.0 0.0 31.4 34.3 97.1 94.1 91.2 60.0 45.0 88.2 79.4 76.5 100.0 0.0 0.0 74.3 0.0 62.9 0.0 94.1 91.2 100.0 37.5 100.0 37.1 100.0 99.0 99.0 98.0 100.0 100.0 100.0 99.0 100.0 100.0 100.0 100.0 92.9 100.0 97.0 94.9 100.0 97.0 100.0 100.0 100.0 100.0 100.0 91.0 5.7 100.0 100.0 100.0 100.0 100.0 97.1 100.0 100.0 94.1 91.2 100.0 26.5 20.0 94.3 5.9 100.0 60.0 100.0 100.0 100.0 100.0 100.0 85.7 37.4 86.0 50.5 84.7 76.5 64.0 92.0 74.0 94.0 90.8 16.2 83.8 86.0 85.7 88.6 97.1 82.4 61.8 10.0 37.1 40.0 91.4 94.1 97.1 0.0 5.7 95.0 100.0 96.0 99.0 100.0 99.0 100.0 94.0 99.0 100.0 93.9 100.0 97.0 100.0 97.1 68.6 91.2 94.1 97.5 97.1 88.6 97.1 100.0 100.0 100.0 94.3

One Step Malaria P.f/Pan Testj OnSite Pf/Pan Ag Rapid Testj OptiMAL-IT ParaHIT - Total Ver. 1.0 (Device) ParaHIT - Total Ver. 1.0 (Dipstick) ParaHIT® total (dipstick) Parascreen® - Rapid test for Malaria Pan/Pfj RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Ag P.f/Panj SD BIOLINE Malaria Ag Pf/ Pan SD BIOLINE Malaria Agj Surestep™ Easy Malaria Pf/Pan Rapid Test Device Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom Advanced Quality™ One Step Malaria P.f/P.v Tri-Line Test Advantage Malaria Card ASAN Easy Test® Malaria Pf/Pan Ag BIONOTE MALARIA P.f.& P.v. Ag Rapid Test Kit CareStart™ Malaria HRP2/pLDH (Pf/Pv) COMBOj CareStart™ Malaria HRP2/pLDH (Pf/VOM) COMBOj Core™ Malaria Pv/Pf diagnosticks- Malaria (Pv/Pf) Cassette FalciVax™ - Rapid test for Malaria Pv/Pfj HiSens Malaria Ag P.f/P.v Combo Card HiSens Malaria Ag P.f/VOM Combo Card Humasis Malaria P.f/P.v Antigen Test Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Malaria Pf/Pv Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Testj Maleriscan® Malaria Pf/Pv Medisensor Malaria HRP2/pLDH (Pf/Pv) COMBO Medisensor Malaria HRP2/pLDH (Pf/VOM) COMBO One Step Malaria P.F/P.V Test (Cassette)j OnSight™ - ParaQuick-2 (Pv,Pf) Malaria Test OnSite Pf/Pv Ag Rapid Testj ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device

W56-C R0113C 710024 55IC204-10 55IC203-10 55IC201-10 50310025 IMPN-C52 05FK60/05FK63 05FK66 05FK40 IMA-T402 412499

RAPID 1-2-3® HEMA CASSETTE MALARIA PF/PV TEST

ITP11003 TC40 IR211025 AM4650-K RG19-12 G0161 G0171 MAL-190022 KMVFC6002 50300025 HR3123 HR3323 AMFV-7025 1-13-101-3 MFV-124V GM006 GM002 MAT-PF/PAN-50 MAT-50 M161 M171 523352 537-25-DB R0112C 55IC402-50 MAL-PFVCAS/25(100)

Table S2 (continued) Panel detection scorea False-positive rates (%) 200 parasites/μL Pf samples Pv samples Pf samples Pv samples Falsepositive non-Pf infectione False-positive Plasmodium spp. Infectioni 2.2 3.5 32.0 (231) 2.1 3.5 (198) 4.0 (199) 24.5 37.0 (198) NA NA NA NA NA NA NA NA NA NA 1.0 NA NA NA NA NA NA NA 0.4 2.2 0.0 13.5 0.0 0.0 2.5 2.5 0.5 1.0 Performance measure Panel detection score for Pf and Pv 200/µL samples False-positive rates against clean-negatives 0.0 0.2 0.5 0.1 1.0 1.1 0.1 0.7 0.0 0.2 0.0 0.5 0.3 0.0 0.2 0.2 0.2 0.0 4 2 4 5 3 3 2 2 5 4 5 3 3 2 2 2 3 2 Recommended WHO procurement criteria ≥ 75% < 10% Invalid rate < 5% of tests conducted 0.3 0.0 13.3 1.5 (399) 0.3 (391) 0.0 (389) 0.0 (399) 0.0 (396) NA NA NA NA NA NA NA NA NA 0.3 NA NA NA NA NA NA NA NA NA NA NA NA 0.0 (137) 0.0 (139) 2.5 0.0 (159) 0.0 (197) 0.0 (196) 0.0 0.0 (199) 1.4 2.9 (69) 0.0 0.0 0.0 0.0 (159) 27.4 (135) 2.9 0.5 0.0 (199) 16.0 (194) 1.5 0.0 0.0 19.4 (67) 2.9

Total false-positive ratesb (%) Clean-negative samples

200 parasites/μL Manufacturer Pf samplesc Pv samplesd Pf samplesc Pv samplesd Falsepositive Pf infectionf Falsepositive non-Pf infectiong Falsepositive Pf infectionh

2000 or 5000 parasites/μL

2000 or 5000 parasites/μL

Product

Catalogue number

Invalid rate (%)l

Round

05FK100 05FK80/05FK83 A03-12-322 W056-C Core Diagnostics SSA Diagnostics & Biotech Systems Unimed International Inc. Zephyr Biomedicals 77.0 61.2 84.0 81.8 16.2 31.0 25.0 22.0 17.2 NA 92.5 NA 100.0 54.3 92.5 82.5 77.5 62.9 92.9 98.0 87.0 96.0 90.9 100.0 100.0 100.0 100.0 100.0 85.7 99.0 100.0 100.0 0 88.6 98.0 99 99.0 100.0 55.9 97.1 92.9 93.9 89.0 93.0 11.4 11.4 45.0 45.0 99.0 99.0 100.0 100.0 94.3 94.3 100.0 100.0

Standard Diagnostics Inc. Standard Diagnostics, Inc. Artron Laboratories Inc. Guangzhou Wondfo Biotech Co. Ltd.

96.9 96.0 88.8 87.0

97.1 95.0 38.2 28.6

100.0 100.0 99.0 98.0

100.0 100.0 100.0 97.1

SD BIOLINE Malaria Ag Pf/ Pf/ Pvk SD BIOLINE Malaria Ag Pf/Pv Trusty™ Malaria Antigen P.f./p.v. test Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pf, Pv and pan Core™ Malaria Pan/Pv/Pf diagnosticks MALARIA (Pan/Pv/Pf) Cassette FirstSign™ - ParaView-3 (Pan+Pv+Pf) Malaria Test Paramax-3 Rapid Test for Malaria Pan/Pv/Pf (device) Pan only Advantage Pan Malaria Cardj AZOG hCG Malaria Detection Test Device CareStart™ Malaria pLDH (PAN)j

MAL-190026 MPNVFC1007.5 2103 CB-25 50320025

IR013025 MPT-124 G0111

Clearview® Malaria pLDHj

diagnosticks MALARIA (Pan) Cassette First Response® Malaria Ag pLDH FirstSign™ - PanCheck (Pan) Malaria Test OnSight™ - PanScreen (Pan) Malaria Test Parabank™ Device - Rapid test for Malaria Panj Pv only SD BIOLINE Malaria Ag Pv Standard Diagnostics, Inc. g

J. Mitra & Co. Pvt. Ltd. AZOG, INC. Access Bio, Inc. Orgenics Ltd. (Inverness Medical 70884025 Innovations) MPNWBC1007.3 SSA Diagnostics & Biotech Systems I12FRC30 Premier Medical Corporation Ltd. 2104 CB-25 Unimed International Inc. 539-25-DB Amgenix International, Inc. 50301025 Zephyr Biomedical Systems

05FK70

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

NA, not applicable

For combination tests, pan or Pv line, only, positive indicates a false-positive non-P. falciparum infection (round 1, n=158; round 2, n=200; round 3, n=198; Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species round 4, n=196; round 5, n=200) Pvom, Plasmodium vivax, ovale and malariae h Pf line positive indicates a false-positive P. falciparum infection (round 1, n=40; a A sample is considered detected only if all RDTs from both lots read by the first technician, round 2, n=80, round 3, n=70; round 4, n=68; round 5, n=70) at minimum specified reading time, are positive i Round 1, n=168; round 2, n=200; round 3, n=200; round 4, n=232 round 5, n=236 b The total number of times a positive result for malaria was generated when it should not have been j Product resubmission, results from most recent round of testing replace previous c Round 1, n=79; round 2, n=100; round 3, n=99; round 4, n=98; round 5, n=100 results. Refer to Table S1. d Round 1, n=20; round 2, n=40; round 3, n=35; round 4, n=34; round 5, n=35 k PDS presented in the table is based on a positive pf test line (either pf-HRP2 or pfe For combination tests, pan or Pv line, only, positive indicates a false-positive non P. falciparum pLDH). For test line-specific results refer to the tables and annexes in the full reports. infection (round 1 n=316; round 2, n=400; round 3, n=396; round 4, n=392; round 5, n=400) f Pf line positive indicates a false-positive P. falciparum infection (round 1, n=80; round 2, l Round 1, n=954; round 2, n=1240; round 3, n=1204; round 4, n=1192;      Round 5, n=1214 n=160; round 3, n=140; round 4, n=136; round 5, n=140)

Summary Rounds 1-5

15

16

Table S3: Malaria RDT rounds 2—5 heat stability results on a cultured P. falciparum sample at low (200) and high (2000) parasite density (parasites/μL). Positivity rate at baseline and after 60 days’ incubation at 35 °C and 45 °C Percentage positive test results for P. falciparum (Pf line) Percentage positive test results for P. falciparum (Pf line) 2000 parasites/μL Baseline Number of tests positive Lots 1 and 2 combined 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 83.3 86.7 0.0 80.0 100.0 73.3 96.7 0.0 90.0 100.0 10.0 100.0 0.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 0.0 3.3 0.0 0.0 0.0 0.0 10.0 0.0 0.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 80.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 65.0 100.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 100.0 100.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 100.0 100.0 NA NA NA

Percentage positive test results for P. falciparum (pan line) 200 parasites/μL Baseline Number of tests positive Lots 1 and 2 combined 35 °C 45 °C Baseline 35 °C 2000 parasites/μL

Percentage positive test results for P. falciparum (pan line) 45 °C Round

Product Baseline 35 °C Number of tests positive Lots 1 and 2 combined ABON Biopharm (Hangzhou) Co. Ltd InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. RapiGen Inc. Bionote,Inc. Access Bio, Inc. Access Bio, Inc. Vision Biotech (Pty) Ltd Core Diagnostics SSA Diagnostics & Biotech Systems SSA Diagnostics & Biotech Systems Premier Medical Corporation Ltd. Unimed International Inc. HBI Co., Ltd. ICT International Biosynex Biosynex IND Diagnostics Inc. Shanghai Kehua Bio-engineering Co.,Ltd. Bhat Bio-Tech India (Pte.) Ltd. Bioland, Ltd Blue Cross Bio-Medical (Beijing) Co., Ltd. Amgenix International, Inc. CTK Biotech, Inc. Orchid Biomedical Systems Orchid Biomedical Systems Span Diagnostics Ltd. Span Diagnostics Ltd. Standard Diagnostics Inc. Standard Diagnostics, Inc. Artron Laboratories Inc. Vision Biotech (Pty) Ltd Guangzhou Wondfo Biotech Co. Ltd. 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.7 100.0 100.0 96.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.0 100.0 100.0 96.7 96.7 100.0 100.0 100.0 100.0 100.0 96.7 100.0 100.0 86.7 100.0 100.0 100.0 96.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 86.7 100.0 100.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 56.7 100.0 100.0 56.7 100.0 100.0 93.3 96.7 90.0 15.0 15.0 17.0

Catalogue number Manufacturer 35 °C 45 °C 45 °C

200 parasites/μL

Number of tests positive Lots 1 and 2 combined NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 70.0 70.0 70.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 90.0 100.0 100.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 30.0 80.0 80.0 4 5 5 4 3 5 2 3 3 5 2 5 4 2 3 3 5 4 5 4 3 4 2 3 4 4 3 3 3 5 4 5 4 3 5 5 5

Pf only ABON™ Malaria P.f. Rapid Test Device (Whole Blood)

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013) ABON Biopharm (Hangzhou) Co. Ltd. ABON Biopharm (Hangzhou) Co. Ltd LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd.

IMA-402 ITP11002TC1/ TC40 Advantage P.f. Malaria Carda IR016025 BIOCREDIT Malaria pf(HRP II) HR0100 BIONOTE MALARIA P.f. Ag Rapid Test Kit RG19-11 CareStart™ Malaria HRP2 (Pf)a G0141 CareStart™ Malaria HRP2/pLDH Pf test G0181 Clearview® Malaria P.f.a VB01 Core™ Malaria Pf MAL-190020 diagnosticks- Malaria (Pf) Cassette WBa KMFC6001 diagnosticks- Malaria (Pf) Dipstick WB KMFD6007 First Response® Malaria Ag P. falciparum (HRP2) Card Testa I13FRC FirstSign™ Malaria Pf 2100CB-25 HiSens Malaria Ag Pf HRP2 Card HR3023 ICT Diagnostics Malaria P.f.a ML01 IMMUNOQUICK CONTACT falciparum 0519K25 IMMUNOQUICK® MALARIA falciparuma 0502_K25 IND ONE STEP MALARIA ANTIGEN P.f 535-11 KHB® Malaria Ag P.f Rapid Test KH-R-06-20 Maleriscan ® Malaria P.f Antigen Test MAT-PF-50 NanoSign Malaria Pf Ag RMAF10 One Step Malaria P.F Test (Cassette)a 522352 OnSight™ - Malaria Pf Test 511-25-DB OnSite Pf Ag Rapid Testa R0114C Paracheck® Pf-Rapid Test for P. falciparum Malaria Device (Ver.3)j 302030025 Paracheck® Pf-Rapid Test for P. falciparum Malaria Dipstick (Ver.3)j 302040025 ParaHIT® - f (Device) 55IC104-50 ParaHIT® -f (Dipstick) 55IC103-50 SD BIOLINE Malaria Ag P.f. (HRP2/pLDH)b 05FK90 SD BIOLINE Malaria Ag Pfa 05FK50/05FK53 Trusty™ Malaria Antigen P.f. test A03-01-322 Vision Malaria Pf VB01 Wondfo One Step Malaria P.f Testa W 37-C Pf and pan ABON Malaria Pan/P.f. Rapid Test Device IMA-B402 ABON™ Plus Malaria P.f/Pan Rapid Test Device (Whole Blood) IMA-T402

Advanced Quality™ One Step Malaria Pf Testa

ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test

MAGC 25

Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Carda

ITP11005 IR221025

Table S3 (continued) Percentage positive test results for P. falciparum (Pf line) Percentage positive test results for P. falciparum (Pf line) 2000 parasites/μL Baseline 45 °C Number of tests positive Lots 1 and 2 combined 80.0 3.3 0.0 0.0 0.0 100.0 93.3 100.0 100.0 0.0 0.0 26.7 0.0 0.0 0.0 0.0 0.0 0.0 3.3 0.0 100.0 0.0 0.0 3.3 0.0 20.0 13.3 10.0 0.0 16.6 13.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 10.0 0.0 0.0 0.0 0.0 100.0 0.0 0.0 20.0 0.0 3.3 53.3 3.3 0.0 0.0 93.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.7 36.7 3.3 0.0 13.3 0.0 13.3 0.0 95.0 0.0 0.0 13.3 0.0 33.3 40.0 0.0 0.0 0.0 100.0 0.0 0.0 6.7 0.0 0.0 0.0 0.0 0.0 73.3 53.3 0.0 0.0 100.0 86.7 100.0 100.0 0.0 10.0 80.0 0.0 0.0 0.0 0.0 66.7 100.0 53.3 100.0 93.3 0.0 3.3 83.3 0.0 0.0 0.0 0.0 20.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 90.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 50.0 0.0 35.0 100.0 100.0 90.0 100.0 50.0 100.0 10.0 100.0 0.0 90.0 60.0 60.0 100.0 100.0 100.0 0.0 100.0 0.0 100.0 70.0 93.3 26.7 100.0 100.0 0.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 20.0 90.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 0.0 0.0 100.0 100.0 90.0 50.0 50.0 100.0 60.0 100.0 0.0 40.0 100.0 90.0 90.0 100.0 100.0 0.0 100.0 0.0 100.0 80.0

Percentage positive test results for P. falciparum (pan line) 200 parasites/μL Baseline 45 °C 35 °C Baseline 35 °C 2000 parasites/μL 45 °C Number of tests positive Lots 1 and 2 combined 100.0 0.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 0.0 100.0 100.0 90.0 80.0 100.0 100.0 100.0 10.0 0.0 0.0 100.0 100.0 90.0 70.0 100.0 100.0 40.0 90.0 0.0 50.0 100.0 50.0 100.0 100.0 100.0 0.0 100.0 0.0 100.0 100.0

Percentage positive test results for P. falciparum (pan line) Round

Product Baseline Number of tests positive Number of tests positive Lots 1 and 2 combined 100.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 20.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 0.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

Catalogue number Manufacturer 35 °C 45 °C 35 °C

200 parasites/μL

Lots 1 and 2 combined J. Mitra & Co. Pvt. Ltd. AZOG, INC. 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.7 100.0 100.0 35.0 100.0 100.0 100.0 96.7 100.0 100.0 60.0 10.0 100.0 100.0 46.7 56.7 100.0 96.7 100.0 100.0 100.0 0.0 100.0 46.7 100.0 100.0 100.0 100.0 0.0 100.0 100.0 100.0 96.7 100.0 100.0 33.3 6.7 100.0 96.7 56.7 23.3 100.0 100.0 100.0 96.7 100.0 0.0 100.0 13.3 100.0 100.0 93.3 43.3 5.0 100.0 100.0 100.0 93.3 100.0 100.0 23.3 0.0 100.0 96.7 66.7 26.7 96.7 96.7 100.0 96.7 100.0 0.0 100.0 26.7 100.0 100.0 100.0 100.0 96.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.7 96.7 90.0 100.0 96.7 100.0 93.3 100.0 100.0 100.0 96.7 96.7 96.7 96.7 100.0 100.0 100.0 100.0

IR231025

5 4 5 3 5 3 5 3 3 3 5 4 3 5 5 5 4 5 5 2 2 5 5 4 3 4 3 5 3 4 5 4 5 3 5 4 4 3 5 3

MFV-124F

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

Advantage Malaria Pan + Pf Card AZOG Malaria pf (HRPII)/pf (LDH)/ (PAN-LDH) Antigen Detection Deviceb BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BIONOTE MALARIA P.f.& Pan Ag Rapid Test Kit BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria/Pregnancy Combo (pLDH/HRP2/HCG) CareStart™ Malaria HRP2/pLDH (Pf/PAN) COMBOa CareStart™ Malaria pLDH 3 Line Test CareStart™ Malaria Screen Clearview® Malaria Comboa Clearview® Malaria Dual Core™ Malaria Pan Pf diagnosticks MALARIA (Pan/Pf) Cassette DIAQUICK Malaria P.f/Pan Cassette

RapiGEN INC. Bionote,Inc. Bio Focus Co., Ltd. Access Bio Inc Access Bio, Inc. Access Bio, Inc. Access Bio, Inc. Vision Biotech (Pty) Ltd Orgenics Ltd.(IS) Core Diagnostics Ltd. SSA Diagnostics & Biotech Systems DIALAB GmbH Advy Chemical (Affiliate of Bharat EzDx™ Malaria Pan/Pf Rapid Test Detection kita RK MAL 001 Serums & Vaccines Ltd. ) First Response® Malaria Ag. pLDH/HRP2 Combo Card Testa I16FRC Premier Medical Corporation Ltd. FirstSign™ ParaView (Pan+Pf)a 2101CB-25 Unimed International Inc. GenBody™Malaria Pf/Pan Ag MALAG100 GenBody Inc. Genedia® Malaria P.f/Pan Ag Rapid Test 20-0146-01 Green Cross Medical Science Corp. (Korea) HiSens Malaria Ag P.f/P.v Card HR2823 HBI Co., Ltd. HiSens Malaria Ag Pf/Pv (HRP2/pLDH) Card HR2923 HBI Co., Ltd. Humasis Malaria P.f/Pan Antigen Testj AMAL-7025 Humasis, Co., Ltd. ICT Malaria Dual Testa ML03 ICT International ICT MALARIA COMBOa ML02 ICT International IMMUNOQUICK CONTACT MALARIA +4 0525K25 Biosynex IND ONE STEP MALARIA ANTIGEN P.f/Pan TEST 535-10 IND Diagnostics Inc. Malaria Pan Test MAL-W23N-001 Dima • Gesellschaft für Diagnostika mbH Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit A03-18-322 Artron Laboratories Inc. Malaria pf (HRP II) / (PAN-pLDH) Antigen Detection Test Devicea MFV-124R AZOG, Inc. Malaria pf (HRP II)/PAN (pLDH) Antigen Detection Test Device 1-13-101-1 United Biotech, Inc. Malaria pf (pLDH) / PAN-pLDH Test Device a MFV-124 AZOG, Inc. Malaria Pf/ PAN GM004 Genomix Molecular Diagnostics Pvt.Ltd. Malaria Pf/Pan One Step Rapid Test RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. Malascan™ Device - Rapid test for Malaria Pf/Pana 50402025 Zephyr Biomedical Systems MD Malaria Pf/Pan(pLDH) test MDMALLDH001 Medical Diagnostech (Pty) Ltd MeDiPro Malaria Ag HRP2/pLDH Combo IR-0051K Formosa Biomedical Technology Corp. NanoSign Malaria pf/pan Ag 3.0a RMAP10 Bioland Ltd. NanoSign Malaria Pf/Pv Ag RMAD10 Bioland, Ltd NG-Test MALARIA Pf/Pan (pLDH) NG-MAL-W23-001 SARL NG Biotech, Z.A. One Step Malaria P.f/Pan Testa W56-C Guangzhou Wondfo Biotech Co. Ltd.

C30RHA25 RG19-08 17012 G0221 G0131 G0121 G0231 VB11 VB20 MAL-190024 MPNFWBC 1007.4 Z11200CE

(continued)

Summary Rounds 1-5

17

18

Table S3: Malaria RDT rounds 2—5 heat stability results on a cultured P. falciparum sample at low (200) and high (2000) parasite density (parasites/μL). Positivity rate at baseline and after 60 days’ incubation at 35 °C and 45 °C (continued) Percentage positive test results for P. falciparum (Pf line) Percentage positive test results for P. falciparum (Pf line) 2000 parasites/μL Baseline Number of tests positive Lots 1 and 2 combined 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.7 100.0 96.7 100.0 36.7 93.3 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 90.0 100.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.0 100.0 100.0 100.0 100.0 100.0 100.0 70.0 100.0 100.0 95.0 100.0 100.0 100.0 85.0 100.0 90.0 100.0 100.0 95.0 100.0 100.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 80.0 100.0 100.0 100.0 0.0 100.0 60.0 95.0 100.0 100.0 100.0 100.0 90.0 100.0 100.0 0.0 0.0 0.0 50.0 10.0 10.0 0.0 0.0 16.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.3 20.0 0.0 10.0 0.0 0.0 0.0 3.3 0.0 0.0 0.0 0.0 13.3 100.0 0.0 0.0 0.0 0.0 0.0

Percentage positive test results for P. falciparum (pan line) 200 parasites/μL Baseline Number of tests positive Lots 1 and 2 combined 90.0 100.0 100.0 100.0 50.0 100.0 100.0 100.0 90.0 80.0 0.0 60.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Percentage positive test results for P. falciparum (pan line) 2000 parasites/μL Baseline 35 °C 45 °C Number of tests positive Lots 1 and 2 combined 100.0 90.0 100.0 90.0 45.0 100.0 60.0 100.0 100.0 20.0 0.0 60.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 100.0 0.0 100.0 0.0 70.0 100.0 100.0 100.0 100.0 90.0 0.0 0.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 5 3 4 4 2 5 5 5 4 3 3 5 3 3 5 3 4 4 3 2 4 4 4 4 4 3 5 4 5 2 4 4 5 2 4 5 4 4 2 4 5

Product Baseline 35 °C 35 °C Number of tests positive Lots 1 and 2 combined CTK Biotech, Inc. Diamed - A Division of Bio-Rad Span Diagnostics Ltd. Span Diagnostics Ltd. Span Diagnostics Ltd Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. Standard Diagnostics Inc. Standard Diagnostics Inc. ACON Biotech (Hangzhou) Co. Ltd. IMACCESS S.A.S InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. ASAN Pharmaceutical Co., Ltd Bionote,Inc. Access Bio, Inc. Access Bio, Inc. Core Diagnostics SSA Diagnostics & Biotech Systems Zephyr Biomedicals HBI Co., Ltd. HBI Co., Ltd. Humasis, Co., Ltd. United Biotech, Inc. AZOG, Inc. Genomix Molecular Diagnostics Pvt. Ltd. Genomix Molecular Diagnostics Pvt.Ltd. Bhat Bio-Tech India (P) Ltd. Bhat Bio-Tech India (P) Ltd Medisensor, Inc. Medisensor, Inc. Blue Cross Bio-Medical (Beijing) Co., Ltd. Amgenix International, Inc. CTK Biotech, Inc. Span Diagnostics Ltd. Hema Diagnostic Systems, LLC Standard Diagnostics Inc. Standard Diagnostics, Inc. Artron Laboratories Inc. Guangzhou Wondfo Biotech Co. Ltd. 96.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 83.3 40.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 96.7 96.7 96.7 100.0 100.0 100.0 95.0 100.0 100.0 100.0 100.0 100.0 100.0 90.0 33.3 100.0 60.0 100.0 100.0 100.0 100.0 100.0 96.7 100.0 96.7 63.3 100.0 100.0 100.0 100.0 95.0 100.0 100.0 100.0 100.0 100.0 96.7 83.3 40.0 100.0 30.0 100.0 100.0 100.0 100.0 90.0 96.7 100.0 0.0 100.0 100.0 55.0 100.0 100.0 100.0 96.7 0.0 100.0 100.0 100.0 0.0 100.0 93.3 85.0 100.0 100.0 100.0 96.7 0.0 100.0 96.7 100.0 0.0 100.0 46.7 55.0 100.0 100.0 100.0 100.0 0.0 100.0 96.7

Catalogue number Manufacturer 35 °C 45 °C 45 °C 45 °C

200 parasites/μL

Round

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

OnSite Pf/Pan Ag Rapid Testa OptiMAL-IT ParaHIT - Total Ver. 1.0 (Device) ParaHIT - Total Ver. 1.0 (Dipstick) ParaHIT® total (dipstick) Parascreen® - Rapid test for Malaria Pan/Pfa RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Ag P.f/Pana SD BIOLINE Malaria Ag Pf/ Pan SD BIOLINE Malaria Aga Surestep™ Easy Malaria Pf/Pan Rapid Test Device Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom Advanced Quality™ One Step Malaria P.f/P.v Tri-Line Test Advantage Malaria Card ASAN Easy Test® Malaria Pf/Pan Ag BIONOTE MALARIA P.f.& P.v. Ag Rapid Test Kit CareStart™ Malaria HRP2/pLDH (Pf/Pv) COMBOa CareStart™ Malaria HRP2/pLDH (Pf/VOM) COMBOa Core™ Malaria Pv/Pf diagnosticks- Malaria (Pv/Pf) Cassette FalciVax™ - Rapid test for Malaria Pv/Pfa HiSens Malaria Ag P.f/P.v Combo Card HiSens Malaria Ag P.f/VOM Combo Card Humasis Malaria P.f/P.v Antigen Test Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device Malaria pf (HRP II) / pv (pLDH) Antigen Detection Test Device Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Malaria Pf/Pv Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test Maleriscan® Malaria Pf/Pv Medisensor Malaria HRP2/pLDH (Pf/Pv) COMBO Medisensor Malaria HRP2/pLDH (Pf/VOM) COMBO One Step Malaria P.F/P.V Test (Cassette)a OnSight™ - ParaQuick-2 (Pv,Pf) Malaria Test OnSite Pf/Pv Ag Rapid Testa ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device

R0113C 710024 55IC204-10 55IC203-10 55IC201-10 50310025 IMPN-C52 05FK60/05FK63 05FK66 05FK40 IMA-T402 412499

RAPID 1-2-3® HEMA CASSETTE MALARIA PF/PV TEST

SD BIOLINE Malaria Ag Pf/ Pf/ Pvb SD BIOLINE Malaria Ag Pf/Pv Trusty™ Malaria Antigen P.f./p.v. test Wondfo® One Step Malaria P.f/P.v Whole Blood Test

ITP11003 TC40 IR211025 AM4650-K RG19-12 G0161 G0171 MAL-190022 KMVFC6002 50300025 HR3123 HR3323 AMFV-7025 1-13-101-3 MFV-124V GM006 GM002 MAT-PF/PAN-50 MAT-50 M161 M171 523352 537-25-DB R0112C 55IC402-50 MAL-PFVCAS/25(100) 05FK100 05FK80/05FK83 A03-12-322 W056-C

Table S3 (continued) Percentage positive test results for P. falciparum (Pf line) Percentage positive test results for P. falciparum (Pf line) 2000 parasites/μL Baseline 45 °C Number of tests positive Lots 1 and 2 combined 0.0 0.0 60.0 100.0 36.7 100.0 100.0 96.7 0.0 50.0 25.0 5.0 0.0 NA 93.3 0.0 80.0 5.0 35.0 0.0 NA 66.7 100.0 100.0 60.0 100.0 100.0 100.0 0.0 55.0 10.0 15.0 0.0 NA 0.0 0.0 50.0 25.0 0.0 0.0 15.0 30.0 80.0 70.0 100.0 100.0 100.0 100.0 100.0 100.0 80.0 100.0 100.0 100.0 90.0 NA

Percentage positive test results for P. falciparum (pan line) 200 parasites/μL Baseline 45 °C 35 °C Baseline 35 °C 2000 parasites/μL 45 °C Number of tests positive Lots 1 and 2 combined 50.0 0.0 90.0 95.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 NA 70.0 50.0 100.0 100.0 90.0 100.0 100.0 100.0 80.0 100.0 100.0 100.0 100.0 NA

Percentage positive test results for P. falciparum (pan line) Round

Product Baseline Number of tests positive Number of tests positive Lots 1 and 2 combined 100.0 100.0 100.0 100.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

Catalogue number Manufacturer 35 °C 45 °C 35 °C

200 parasites/μL

Lots 1 and 2 combined Core Diagnostics SSA Diagnostics & Biotech Systems Unimed International Inc. Zephyr Biomedicals NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 100.0 96.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0 93.3 100.0 100.0

Pf, Pv and pan Core™ Malaria Pan/Pv/Pf diagnosticks MALARIA (Pan/Pv/Pf) Cassette FirstSign™ - ParaView-3 (Pan+Pv+Pf) Malaria Test Paramax-3 Rapid Test for Malaria Pan/Pv/Pf (device) Pan only Advantage Pan Malaria Carda AZOG hCG Malaria Detection Test Device CareStart™ Malaria pLDH (PAN)a

MAL-190026 MPNVFC1007.5 2103 CB-25 50320025

3 3 2 2 5 4 5 3 3 2 2 2 3 2

IR013025 MPT-124 G0111

Clearview® Malaria pLDHa

diagnosticks MALARIA (Pan) Cassette First Response® Malaria Ag pLDH FirstSign™ - PanCheck (Pan) Malaria Test OnSight™ - PanScreen (Pan) Malaria Test Parabank™ Device - Rapid test for Malaria Pana Pv only SD BIOLINE Malaria Ag Pv Standard Diagnostics, Inc.

J. Mitra & Co. Pvt. Ltd. AZOG, INC. Access Bio, Inc. Orgenics Ltd. (Inverness Medical 70884025 Innovations) MPNWBC1007.3 SSA Diagnostics & Biotech Systems I12FRC30 Premier Medical Corporation Ltd. 2104 CB-25 Unimed International Inc. 539-25-DB Amgenix International, Inc. 50301025 Zephyr Biomedical Systems

05FK70

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae

a

Indicates results for those products that meet all WHO recommended procurement criteria Product resubmission, results from most recent round of testing replace previous results. Refer to Table S1. b Results presented in the table are based on stability of a Pf test line (either Pf-HRP2 or Pf-pLDH). Results based on stability of individual test lines is presented in the following table: Percentage positive test results Percentage positive test results Percentage positive test results Percentage positive test results for P. falciparum (Pf line) for P. falciparum (pan line) for P. falciparum (pan line) for P. falciparum (Pf line) 200 parasites/μL Baseline Number of tests positive Lots 1 and 2 combined 100.0 100.0 100.0 0.0 0.0 0.0 96.7 13.3 100.0 26.7 100.0 3.3 3.3 96.7 100.0 6.7 96.7 3.3

Product 35 °C 45 °C Baseline

Catalogue number

Manufacturer

2000 parasites/μL 35 °C 45 °C Number of tests positive Lots 1 and 2 combined 100.0 100.0 100.0 33.3 33.3 33.3 100.0 50.0 100.0 100.0 100.0 10.0 100.0 100.0 100.0 50.0 100.0 100.0

200 parasites/μL Baseline 35 °C 45 °C Number of tests positive Lots 1 and 2 combined NA NA NA NA NA NA 3.3 3.3 NA NA 0.0 0.0 NA NA 0.0 0.0 NA NA

2000 parasites/μL Baseline 35 °C 45 °C Number of tests positive Lots 1 and 2 combined NA NA NA NA NA NA 20.0 20.0 NA NA 0.0 0.0 NA NA 0.0 0.0 NA NA

Round

05FK90 05FK90

Standard Diagnostics Inc. Standard Diagnostics Inc.

NA NA 4 4 4 4

MFV-124F

AZOG, INC.

Malaria Rapid Diagnostic Test Performance – Summary results of WHO product testing of malaria RDTs: Round 1-5 (2008-2013)

SD BIOLINE Malaria Ag P.f. (HRP2/pLDH) - (PF(HRP2) line) SD BIOLINE Malaria Ag P.f. (HRP2/pLDH)- (PF(pLDH) line) AZOG Malaria pf (HRPII)/pf (LDH)/ (PAN-LDH) Antigen Detection Device - (PF(HRP2) line) AZOG Malaria pf (HRPII)/pf (LDH)/ (PAN-LDH) Antigen Detection Device - (PF(pLDH) line) SD BIOLINE Malaria Ag Pf/ Pf/ Pv - (PF(HRP2) line) SD BIOLINE Malaria Ag Pf/ Pf/ Pv - (PF(pLDH) line)

MFV-124F

AZOG, INC.

05FK100 05FK100

Standard Diagnostics Inc. Standard Diagnostics Inc.

Summary Rounds 1-5

19

2. WHO MALARIA RDT PRODUCT TESTING: Round 5: Executive Summary 2.1. Introduction WHO estimates that half the world’s population is at risk of malaria. In 2012, there were an estimated 207 million cases (with an uncertainty range of 135 million to 287 million) and an estimated 627 000 deaths (with an uncertainty range of 473 000 to 789 000). Approximately 90% of all malaria deaths occur in sub-Saharan Africa, and 77% occur in children under 5 years. Malaria remains endemic in 104 countries, and, while parasite-based diagnosis is increasing, most suspected cases of malaria are still not properly confirmed, resulting in over-use of antimalarial drugs and poor disease monitoring (1). WHO recommends that malaria case management be based on parasite diagnosis in all cases (2). The use of antigendetecting rapid diagnostic tests (RDTs) is a vital part of this strategy, forming the basis for extending access to malaria diagnosis by providing parasite-based diagnosis in areas where good-quality microscopy cannot be maintained.The data generated by the WHO and FIND programme to evaluate and compare the performance of commercially available malaria RDTs are guiding procurement decisions, which, in turn, have shifted markets towards better performing tests and helped to improve the quality of manufacturing. The results of WHO malaria RDT product testing form the basis of procurement criteria and constitute the laboratory evaluation component of WHO prequalification for malaria RDTs. This report provides the results of round 5 of product testing, performed at the CDC in 2013, with data on the performance of 42 products. This evaluation adds to the evaluations of rounds 1–4 (3–6), which should be considered as a single evaluation, except that the results for products tested in previous rounds that were resubmitted for testing replace those reported previously. From round to round, the evaluation panels are essentially the same (Annex S1), and the same or slightly modified testing protocols are followed. This report extends the data from previous rounds and therefore increases the number of RDTs available for procurement for which detailed, comparative data are available on aspects of performance relevant to field use. The report provides updated data on the performance of products at least every 5 years, as a result of implementation of the compulsory resubmission policy. Products that are not resubmitted are deleted from the summary results of WHO product testing and are not eligible for WHO procurement.

2.2. The WHO product testing programme Product testing is part of the WHO-FIND malaria RDT evaluation programme, which develops methods for evaluation and provides data on antigen-detecting malaria RDTs. The programme is a collaboration among many institutions in malaria-endemic and non-endemic countries, with a global specimen bank and testing performed at the CDC (Fig. 2). All companies that manufacture according to ISO 13485:2003 quality system standards were invited to submit one test for evaluation in the programme, except manufacturers who were required to submit additional products under the new compulsory resubmission rules. The 42 products from 34 manufacturers1 were evaluated with prepared blood panels of cultured P. falciparum parasites, patientderived wild-type P. falciparum and P. vivax parasites, and a parasite-negative panel. Thermal stability was assessed after 2 months of storage at elevated temperature and humidity, and a descriptive assessment of ease of use was recorded. As for previous rounds, RDTs are grouped in the tables and figures into those that detect P. falciparum only, various combination tests and those that have only a pan-specific (or P. vivax-specific) line. Manufacturers submitted two lots of each product for evaluation. The 23 products that had been tested in previous rounds comprised 10 compulsory resubmissions and 13 voluntary resubmissions (Table 1a,b). The aim of the evaluation is to provide comparative data on the performance of the submitted production lots of each product against samples containing low (200 parasites/µL) and high densities (2000 or 5000 parasites/µL) of P. falciparum or P. vivax . Because the concentration of target antigens in samples with the same parasite density is variable, the evaluation panel selection process is adjusted to ensure that there is no statistically significant difference in mean or median antigen concentrations for HRP2, aldolase and pLDH between panels used in different rounds of testing (Annex S1, Table 3). The data in this report are used to guide procurement decisions by WHO, other United Nations agencies and national governments. Product testing is part of a continuing programme to improve the quality of RDTs that are used and to support widespread, reliable malaria diagnosis in areas where malaria is prevalent. A sixth round of product testing began in June 2014, and the results will be published in 2015.

1

Several manufacturers are subsidiaries of Alere™ .

20

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

2.3. Results of the evaluation The results (summarized in Tables 4–6 and Figs 10, 11, 14 and 15) provide a comparison of two lots of products against a panel of parasite samples diluted to a low parasite density (200 parasites/µL), considered to be close to the threshold that tests must detect in order reliably to identify clinical malaria in many settings (9), and a higher parasite density (2000 or 5000 parasites/µL). For the purposes of this report, the main measure of performance is the PDS, the percentage of malaria samples in a panel that give a positive result in two RDTs per lot at the lower parasite density and a single RDT per lot at the higher parasite density. Thus, it is not a measure of clinical sensitivity or of the positivity rate against the panel but rather a combined measure of positivity rate and inter-test and inter-lot consistency. As for products evaluated in previous rounds of product testing, the PDS varies widely, some products showing high performance in detecting parasites, in thermal stability and other performance measures. Overall, there is no obvious trade-off between the PDS (or positivity rate) and the false-positive rate, which are surrogates for sensitivity and specificity in the field, respectively. Furthermore, a number of tests showed good outcomes for both these indicators. The basis for P. falciparum detection by combination RDTs (P. falciparum/pan, P. falciparum/P. vivax, P. falciparum/P. vivax, ovale and malariae), particularly in samples with low parasite density, is predominantly detection of HRP2 and not pLDH. In other words, mainly the HRP2 test band reacts with P. falciparum-containing samples, probably reflecting poorer affinity of the monoclonal pLDH antibodies on the pLDH test band and not HRP2 persistent antigenaemia, as all samples are known to contain P. falciparum (and pLDH). In round 5, the results for 80% (8/10) and 75% (3/4) of the compulsorily retested products were within 10% of the initial test for detection of P. falciparum and P. vivax at 200 parasites/µL. Most of the differences detected were decreases in performance in comparison with previous testing. Among the voluntary resubmissions, 77% (10/13) and 73% (8/11) of products showed better detection of P. falciparum and P. vivax at 200 parasites/µL, respectively. Detection of P. falciparum improved by 11% on average, while the mean improvement in P. vivax detection was 29%. Two products, however, showed significantly increased (> 75%) false-positive rates in round 5. In combination tests, there was no significant correlation between improvements in P. falciparum and P. vivax detection, suggesting that changes in detection of these two parasite species occurred independently of each other. Several products had very high false-positive rates for P. falciparum against clean negatives, and, as previously reported, high false-positive rates were seen with several products against blood samples containing specific immunological abnormalities (e.g. rheumatoid factor, anti-mouse antibodies) (Table A4.9). The number of samples evaluated was, however, small, and the clinical significance of these

results is limited, although they may be important in certain populations with very low parasite prevalence. Some products showed variation in performance between the two lots evaluated, confirming the advisability of lot-testing before field use. Heat (thermal) stability varied widely, some products retaining high positivity rates after 2 months’ storage at 45 °C and 75% humidity. For many products, pan-line performance at baseline and post-heat stress for detection of the P. falciparum isolate were poor and nearly universally poor against low parasite density samples, making true stability difficult to assess. The majority of the products evaluated showed a variable frequency of anomalies that could interfere with test interpretation, the most common being a red background and incomplete clearing (Table 8). The clinical sensitivity of an RDT, i.e. the proportion of people known to have the disease who test positive for it, is highly dependent on local conditions, including the parasite density in the target population, and therefore varies among populations with differing levels of transmission. The results in this report show comparative performance between RDTs, and give an indication of which products are likely to provide higher sensitivity in the field, particularly in populations with low-density infections. In general, as countries reduce malaria prevalence and even move towards malaria elimination, detection of low parasite densities becomes increasingly important in case management. As the PDS at 2000 parasites/ µL indicates, the sensitivity of many of these products will be similar in populations with higher parasite densities, although a subset of any population will include vulnerable individuals who may develop illness at low parasite densities (e.g. young children, pregnant women, those well protected by bed nets) and must always be taken into account when interpreting RDT results. For areas where significant non-expression of HRP2 is known, the results of HRP2-detecting tests in this report should not be considered to predict sensitivity in the field. Only tests targeting P. falciparum by detection of pLDH or aldolase should be considered. Heat stability (summarized in Table 6) is vital to maintaining the sensitivity of a test in the field. For procurement, therefore, the results for stability should be used to ensure that products to be used in areas with high temperatures during transport and storage have demonstrated good stability in the product testing programme. The requirements vary by country; for example, if tests are to be used in areas where the temperature rarely rises above 30 °C, stability at high temperatures is less important. The requirements for ease of use depend on the extent of training and the work environment of users. Particularly in primary health care settings, the simpler the test, the easier it should be to avoid errors in preparation and interpretation.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Executive Summary Round 5 21

2.4.  Use of the results Box 3 outlines WHO’s minimum criteria for selecting RDTs, and tabular results in Tables S2 and 5 are colour-coded to reflect achievement of these requirements, and a webbased tool that allows filtering of product testing results by various parameters to assist in selecting products with the performance characteristics most suitable for a country’s health programme is available and maintained by FIND (12). Annex S3 provides a step-by-step approach to selecting an RDT, taking into consideration local conditions of malaria

transmission and illness (e.g. Plasmodium species, target antigen variation, parasite densities, climate), and other important considerations, including ease of use in the field, for training and retraining and lot testing. RDTs must not be procured without programme and infrastructure preparation for proper use, including supply chain management, training in test usage and disposal, and training in patient management in response to results. Comprehensive guidance on several aspects of procurement can be found in Good practices for selecting and procuring rapid diagnostic tests for malaria and guidance on implementation in Universal access to malaria diagnosis (13, 14).

3. Background During the past decade, new opportunities for the control of malaria have emerged, including use of long-lasting insecticidal nets, indoor residual spraying of insecticides and artemisinin-based combination therapy. These have been shown to reduce the burden of malaria infection in countries where they are adequately implemented. Therefore, the proportion of febrile episodes attributable to malaria is likely to decrease substantially. Despite WHO’s recommendation for a parasitologically confirmed diagnosis of malaria infection before treatment in all cases (2), diagnoses are still often made on clinical grounds (9), whereas in most endemic areas malaria accounts for a minority of cases of “malaria-like” febrile illness. Microscopy has been the cornerstone of diagnosis and is recommended for malaria diagnosis when its quality can be maintained; however, the need for trained personnel and adequate reagents and equipment limits its availability and accessibility in malaria-endemic areas. Rapid, accurate, accessible diagnostic tools are increasingly required as programmes extend parasite-based diagnosis and the prevalence of malaria decreases. RDTs to detect Plasmodium -specific antigens (proteins) in whole blood of infected people have emerged as an attractive alternative to microscopy. The currently available RDTs come in various formats (dipstick, cassette or hybrids) and contain antibodies bound to specific antigens, such as HRP2 specific to P. falciparum, pan-specific and species-specific pLDH or aldolase specific to all the major Plasmodium species (P. falciparum, P. vivax , P. malariae, P. ovale) (Fig. 1).

Figure 1: Mode of action of antigen-detecting malaria RDTs a Bound Ab Lysing agent and labelled Ab Control line (bound Ab)* Test line (bound Ab)*

Mode of action of common malaria RDT format: (a) Dye-labelled antibody (Ab), specific for the target antigen, is present on the lower end of the nitrocellulose strip or in a well provided with the strip. Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line, and either antibody specific for the labeled antibody, or antigen (Ag), is bound at the control line. (b) Blood and buffer, which have been placed on the strip or in the well, are mixed with the labelled antibody and are drawn up the strip across the lines of bound antibody. (c) If antigen is present, some labelled antibody will be trapped on the test line. Other labelled antibody is trapped on the control line.

Free labelled Ab

Nitrocellulose strip *Not normally visible

Bu er/ ushing agent

b Parasite Ag captured by labelled Ab

Parasitized blood

c

Blood and labelled Ab ushed along strip

Labelled Ab–Ag complex captured by bound Ab of test band

Labelled Ab captured by bound Ab of control band

Captured Ag–labelled Ab complex

Captured labelled Ab

22

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

The number of RDTs available on the market has grown rapidly since their introduction in the late 1990s, with an estimated 60 brands and over 200 tests commercially available today and 205 million tests or more procured in 2012 (1). Regulatory control of diagnostics is, however, often weak, and procurement agencies have had considerable difficulty in selecting appropriate RDTs and ensuring their quality. In view of the inconsistency in the results of field studies and the inherent difficulties in assessing large numbers of products in a

The reports of the previous four rounds of product testing have been released annually since 2009 (3–6 ). This fifth report adds data on the performance of 19 new products and updated data on 23 resubmitted RDTs. Testing for round 5 was conducted against an evaluation panel with similar characteristics to previous panels in terms of overall antigen concentration, parasite origin and parasite-negative blood samples (Annex S1). Most panel samples were retained from previous rounds, with 36 of 100 P. falciparum, 7 of 35 P. vivax and 48 of 100 negative samples replaced (new) in round 5.

Malaria, countries or areas at riskand of transmission, 2009 Figure 2: Network of specimen collection, characterization testing sites

HTD CDC DRM EHNRI KEMRI IHRDC IPM IPC RITM

CIDEIM IMT

UCAD

IPB UL

QIMR Collection and testing site Specimen characterization Global specimen bank Countries or areas where malaria transmission occurs Countries or areas with limited risk of malaria transmission No malaria This map is intended as a visual aid only and not as a definitive source of information about malaria endemicity. Source: © WHO 2010. All rights reserved.

CDC, Centers for Disease Control and Prevention (Atlanta, United States of America); CIDEIM, Centro Internacional de Entrenamiento y Investigaciones Médicas (Cali, Colombia); DMR, Experimental Medicine Research Division (Department of Medical Research, Yangon, Myanmar); EHNRI, Ethiopian Health and Nutrition Research Institute (Addis Ababa, Ethiopia); HTD, Hospital for Tropical Diseases (London, United Kingdom); IHRDC, Ifakara Health Research and Development Center (Bagamoyo, United Republic of Tanzania); IMT, Instituto de Medicina Tropical (Universidad Peruana Cayetano Heredia, Lima, Peru); IPB, Institut Pasteur de Bangui (Bangui, Central African Republic); IPC, Institut Pasteur du Cambodge (Phnom Penh, Cambodia); IPM, Institut Pasteur de Madagascar (Antananarivo, Madagascar); KEMRI, Kenya Medical Research Institute (Kisumu, Kenya); QIMR, Queensland Institute of Medical Research (Brisbane, Australia); RITM, Research Institute of Tropical Medicine (Manila, Philippines); UCAD, Université Cheikh Anta DIOP (Dakar, Senegal); UL, University of Lagos (Lagos, Nigeria).

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Executive Summary Round 5 23

To be widely useful, an RDT must be highly sensitive to ensure detection of all clinically significant malaria infections, highly specific to allow monitoring of low malaria prevalence and appropriate management of non-malarial fevers and highly stable to allow transport and storage in ambient conditions in malaria-endemic areas. Published field trials of RDTs show highly variable performance, probably due to poor manufacturing quality, incorrect storage and handling, poor preparation and interpretation, and sometimes poor study methods, analysis and reporting (15–23). In general, diagnostic testing by microscopy or RDT to a level of 200 parasites/µL will reliably detect nearly all clinically relevant infections in malaria-endemic areas (9).

standardized way in field trials, WHO and partners embarked on a programme to evaluate RDTs for malaria in 2002 to ensure standardized assessment of performance and to guide procurement decisions and regulatory mechanisms. Between 2003 and mid-2012, the programme was managed by WHO and TDR in partnership with FIND. After TDR withdrew its involvement in 2012, the WHO Global Malaria Programme assumed a coordinating role. A steering committee oversees the development of and modifications to standard operating procedures (24,25 ). A network of specimen collection sites has been established to provide specimens for a global bank at the CDC and to facilitate local quality control (Fig. 2).

4. Objective The objective of the programme is to evaluate malaria RDTs for performance to guide procurement of RDTs for use in the field in malaria-endemic countries.

5. Materials and methods 5.1. Test selection In August 2012, the WHO-FIND malaria RDT evaluation programme issued a call for expressions of interest to manufacturers of malaria RDTs with information on the requirements for submission of a product to round 5 and the conditions for participation in the evaluation programme (26). Manufacturers of products that had not been retested since round 1 were informed of a new requirement to resubmit products within less than 5 years or to have these products and their performance characteristics removed from the summary results document, which is a compilation of the results of all previous rounds of testing. Other standard requirements included valid ISO 13485:2003 certification from all manufacturing sites, a supply of sufficient quantities of products (1100 tests from each of two lots),1 compliance with the product definition2 and deadlines for document submission. Thirty-nine manufacturers, proposing 99 products, responded to the call. In order to keep to the schedule and budget and to accommodate 10 compulsory product resubmissions, manufacturers were asked to limit their voluntary submissions to one product. Finally, 42 products were tested in round 5 (Table 1a). Catalogue numbers and verification with 1

Manufacturers were requested to supply an additional 500 RDTs per lot voluntarily to support the WHO-FIND evaluation of malaria recombinant antigens. A working definition of a product can be found in Annex 2 (http:// www2.wpro.who.int/NR/rdonlyres/EEF2F4B0-5863-438A-84428F8CC1AA7F8B/0/EOIAnnex1_2_3_Round5_version21.docx , accessed 23 May 2014).

2

Table 1a: Manufacturers and products accepted into round 5 of WHO malaria RDT product testing Programme Manufacturer Product name CareStart™ Malaria pLDH (PAN)b Access Bio. Inc. CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combob CareStart™ Malaria HRP2 (Pf)b Catalogue numbera G0111/G0111-ET G0131/G0131-ET G0141/G0141-ET RK MAL 001 A03-18-322 AM4650-K MFV-124 MAT-PF/PAN-50 17012 0502_K25 523352 Target antigens pan-pLDH pan-pLDH HRP2 pan-pLDH pan-pLDH — HRP2 — HRP2 HRP2

Advy Chemical Pvt. Ltd. (Affiliate EzDx™ Malaria Pan/Pf Rapid Test Detection Kitd of Bharat Serums & Vaccines Ltd. ) Artron Laboratories Inc. ASAN Pharmaceutical Co., Ltd AZOG, INC. Bhat Bio-Tech India (P) Ltd. Bio Focus Co., Ltd. BIOSYNEX Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit ASAN Easy Test® Malaria Pf/Pan Ag Malaria pf-LDH/PAN-LDH Antigen Test Deviced Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Testd BioTracer™ Malaria Pf/PAN Rapid Card IMMUNOQUICK® MALARIA falciparum

Pvom-pLDH HRP2 pan-pLDH PfpLDH

Pvom-pLDH HRP2 pan-pLDH HRP2 Pv-pLDH HRP2 — HRP2

Blue Cross Bio-Medical (Beijing) One Step Malaria P.F/P.V Testd Co., Ltd.

24

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Table 1a: (continued) Manufacturer CTK Biotech Inc. DIALAB GmbH GenBody Inc. Product name OnSite Pf/Pan Ag Rapid Testd DIAQUICK Malaria P.f/Pan Cassette GenBody™ Malaria Pf/Pan Ag Catalogue numbera R0113C Z11200CE MALAG100 GM006 20-0146-01 W056-C IMPN-C52 AMAL-7025 ML03 412499 ITP11005 ITP11002TC1/TC40 IR221025 IR231025 IR013025 IR016025 MAGC 25 MDMALLDH001 Cardb Testb Target antigens HRP2 pan-pLDH pan-pLDH HRP2 pan-pLDH Pv-pLDH aldolase pan-pLDH HRP2 HRP2 pan-pLDH HRP2 pan-pLDH pan-pLDH pan-pLDH HRP2 pan-pLDH HRP2 pan-pLDH HRP2 HRP2 Pv-pLDH HRP2 HRP2 HRP2 HRP2 pan-pLDH aldolase HRP2 — Pf-pLDH HRP2 — — HRP2 pan-pLDH pan-pLDH — HRP2 HRP2 — HRP2 — — HRP2 pan-pLDH — HRP2 pan-pLDH

Genomix Molecular Diagnostics Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Pvt. Ltd. Device Green Cross Medical Science Corp. Genedia® Malaria P.f/Pan Ag Rapid Test (Korea) Guangzhou Wondfo Biotech Co. Wondfo® One Step Malaria P.f/P.v Whole Blood Test Ltd. Hangzhou Biotest Biotech Co. Ltd. RightSign™ Malaria P.f./Pan Rapid Test Cassette Humasis Co., Ltd. ICT INTERNATIONAL IMACCESS S.A.S InTec Products, Inc. Humasis Malaria Pf/Pan Antigen Testd ICT Malaria Dual Testd Vikia® Malaria Ag Pf/Pan Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advanced Quality™ One Step Malaria Pf Advantage Mal J. Mitra & Co. Pvt. Ltd. Cardb

Advantage Malaria Pan + Pf Card Advantage Pan Malaria Avantage P.f Malaria Cardb

LAB-CARE Diagnostics (India) PVT. LTD. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. Premier Medical Corporation RapiGEN INC. Shanghai Kehua Bioengineering Co.,Ltd. Span Diagnostics Ltd.

ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MD Malaria Pf/Pan-pLDH test NG-Test MALARIA Pf/Pan (pLDH) First Response® Malaria Ag P. falciparum (HRP2) Card Testb First Response® Malaria Ag. pLDH/HRP2 Combo Card Testd BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) KHB® Malaria Ag P.f Rapid Test ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Pfb Pf/Pand

NG-MAL-W23-001 HRP2 I13FRC I16FRC C30RHA25 KH-R-06-20 55IC402-50 KMFC6001 05FK50/05FK53 05FK60 VB20 VB01 Pan/Pfd 50310025 RT 20222 HRP2 pan-pLDH pan-pLDH HRP2 Pv-pLDH HRP2 HRP2 pan-pLDH HRP2 HRP2 pan-pLDH HRP2

SSA Diagnostics & Biotech Systems diagnosticks-Malaria (Pf) Rapid Diagnostic Testd Standard Diagnostics Inc.c Orgenics Ltd.(IS)c Vision Biotech (Pty) Ltdc Zephyr Biomedicals SD BIOLINE Malaria Antigen SD BIOLINE Malaria Antigen Clearview® Malaria Vision Malaria Pfd Duald

Parascreen® Rapid test for malaria

Zhejiang Orient Gene Biotech Malaria Pf/Pan One Step Rapid Test Co., Ltd.

Pf, P. falciparum  Pv, P. vivax  Pvom, P. vivax, ovale, malariae  HRP2, histidine-rich protein 2  pLDH, Plasmodium lactate dehydrogenase a The same products may have different catalogue numbers to reflect box sizes, kit contents or site of manufacture. Usually this involves the end portion of the product code. Please contact manufacturers for details b Indicates products submitted for compulsory retesting in round 5 c Alere subsidaries d These products have been submitted voluntarily to previous rounds of WHO malaria RDT product testing (round 1-4). For details on all product resubmissions refer to Table S1.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Methods 25

manufacturers showed that 23 of the 42 products (55%) had been submitted previously to one or more rounds, including 10 (45%) scheduled for compulsory resubmission (Table1b). All the products met the minimum performance requirements1 in the initial evaluation against the P. falciparum culturederived panel (phase 1) and were therefore evaluated fully. Of the 42 products that were fully evaluated, 9 are designed to detect P. falciparum alone, 31 to detect and differentiate P. falciparum from non-P. falciparum malaria and from P. falciparum and P. vivax or P. vivax, ovale, malariae, and 2 to detect P. falciparum and non-P. falciparum malaria without distinguishing between them. Annexes 1 and 2 give a comprehensive overview of the product characteristics.

5.2. The product testing protocol The testing process is outlined in Fig. 3 and in the Methods manual for product testing of malaria rapid diagnostic tests, version 5 (24). In brief, RDTs from each of two lots of each product were evaluated against a panel of parasite-positive and parasite-negative cryopreserved blood samples and a panel of parasite-negative samples. Both lots were also tested for heat (thermal) stability, evaluated after 2 months’ storage at 4 °C, 35 °C and 45 °C. An ease-of-use description was completed in a standard assessment format, and common RDT anomalies were recorded. The testing and all the results were monitored by the WHO-FIND steering committee, and manufacturers were given 30 days to comment on the results for individual products before publication.

1

PDS > 80% against high-density (2000 parasites/µL) P. falciparum in culture

Table 1b: Products due for compulsory resubmission in round 5 Manufacturer Product name CareStart™ Malaria pLDH (PAN) Access Bio, Inc. CareStart™ Malaria HRP2/pLDH (Pf/PAN) COMBO CareStart™ Malaria HRP2 (Pf) Acon Laboratories, Inc Amgenix International, Inc. Biosynex Diagnostics Automation/Cortez Diagnostics Inc. Human GmbH IND Diagnostic Inc. Innovatek Medical Inc. Intec Products, Inc. Malaria Plasmodium falciparum Rapid Test Device (Whole Blood) OnSight - ParaQuick (Pan, Pf) Test Immunoquick Malaria Falciparum Immunoquick Malaria +4 Malaria P.F/Vivax Hexagon Malaria Hexagon Malaria Combi One Step Malaria Antigen Strip Quickstick Malaria Antigen Testb ITP11002TC1 IN660050 IR016025 IR013025 IR221025 II3FRC 25989 05FK50 2102CB-25 Catalogue number G0111 G0131 G0141 IMA-402 536-25DB 0502_K25 0506_K25 172110P-25 58051 58024 820-1 Participation in round 5a Yes Yes Yes No No Yes No No No No No No Yes No Yes Yes Yes Yes No Yes No No

ADVANCED QUALITY TM MALARIA (p.f) POCT

Inverness Medical Innovations, Inc. Binax Now Malaria Advantage P.f. Malaria Card J. Mitra & Co. Pvt. Ltd Advantage Pan Malaria Card Advantage Mal Card Premier Medical Corporation Ltd. Span Diagnostics Standard Diagnostics Unimed International a b

First Response® Malaria Ag HRP2 Parahit-Total Device Rapid Test for P. falciparum and Pan malaria species SD Bioline Malaria Ag Pf FirstSign – Malaria Pf Card Test FirstSign – ParaView-2 (Pv + Pf) Card Test

The results of the first tests of the products in this list that were not retested in round 5 have been removed from tables S2 and S3 and figs S1 and S2. Co-listed with IND Diagnostics - One Step Malaria Antigen Strip (820-1)

26

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Figure 3: Overview of malaria RDT product testing

RDT product testing flow chart

Panel detection score and false-positive rate Test RDTs against high and low density samples of phase 1 panel Proceed to test RDTs against phase 2 panel, if pass phase 1 In each case, read each result with: Place in a 35°C incubator

Heat stability Select RDTs from 2 differents lots

Ease-of-use description

Blood safety Total time to obtain result Number of timed steps Quality of the instructions

Place in a 45°C incubator

Initial test (room temperature) 200, 2000 p/µL

Store for 2 months at 75% humidity

Technician 1

Technician 2

Remove and allow RDTs to reach room temperature

• format • blood transfer method • items included in package • language • anomalies

Additional information

Completed assessment forms

Record results

Prepare RDTs with a 200 p/µL sample

Prepare RDTs with a 2000 p/µL sample

Record results

Record results

5.3. Evaluation panels RDTs were evaluated against three panels: • P. falciparum culture lines (includes a subset, “manufacturer’s panel”) at low (200 parasites/µL) and high parasite density (2000 parasites/µL); • wild-type Plasmodium species (P. falciparum, P. vivax ) from naturally infected humans diluted with parasitenegative samples to low (200 parasites/µL) and high parasite density (2000 or 50001 parasites/µL). All samples are prepared from isolates that express HRP2; and • a parasite-negative panel (“clean” samples and diseasespecific or blood factor-specific samples). An overview of sample collection and characterization is given in the methods manuals prepared for this purpose (24, 25 ). Characterization results for each round are available on the WHO/GMP and FIND websites (27 ). Thus, each panel specimen was characterized for: • species, by duplicate microscopy (two microscopists) and confirmation of mono-species infection by nested polymerase chain reaction (PCR);

• antigen concentration, by quantitative ELISA for HRP2, pLDH and aldolase; and • the absence of malaria parasites by nested PCR and confirmatory testing for other diseases in the case of parasite-negative samples. Most P. falciparum samples in the global specimen bank are also characterized according to HRP2 sequence by PCR amplification and sequencing. This is not performed on samples collected after 2009, as accumulated evidence indicates that HRP2 variation has no significant effect on RDT sensitivity (28). The geographical origin of all samples is recorded.

Panel composition P. falciparum-cultured parasites panel Twenty culture-adapted strains of P. falciparum from various geographical locations were selected, including 14 strains with type B HRP2 sequence, five with type A and one with type C. All specimens were derived from the CDC culture bank and diluted in O-positive blood from donors in the USA (24).

Wild-type parasite panel 1

Four (4%) of the 100 P. falciparum dilution sample sets contained 200 and 5000 parasites/µL, and two (6%) of the 35 P. vivax dilution sample sets contained 200 and 5000 parasites/µL.

The parasite-positive wild-type (clinical) panel consisted of samples from 100 cases of P. falciparum and 35 cases of P. vivax malaria, from 12 collection sites in Africa, Asia and

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Methods 27

South America (Fig. 2, 4a and 4b). Samples were collected from febrile patients and processed by standardized methods designed to preserve the target antigen concentration (25). After dilution and cryopreservation, the samples were transferred to the global bank (WHO specimen bank) at CDC for further characterization. Sample antigen (HRP2, pLDH, aldolase) concentrations determined by quantitative ELISA are given in Table 3. The results are based on 94 P. falciparum samples for pLDH, 99 P. falciparum samples for HRP2 and aldolase, 34 P. vivax samples for pLDH and 35 P. vivax samples for aldolase. This panel is highly comparable to those of previous rounds (Annex S1).

Table 2: Characteristics of Plasmodium spp. negative samples Nature of negative samplea Clean-negativeb Anti-nuclear antibody positive (sera) Anti-mouse antibody positive (plasma) Rheumatoid factor positive (whole blood and sera) Rapid plasma reagin positive (sera) Chagas' disease antibody positive (plasma) Dengue antibody positive (whole blood sera) Leishmaniasis antibody positive (sera) Schistosomiasis antibody positive (whole blood and sera) a

No. 58 13 3 4 5 2 4 5 6

Negative blood sample panel The negative panel consisted of 59 “clean” parasite-negative samples from donor-derived blood obtained in banks or from volunteers in non-endemic (USA) and endemic areas (Kenya, the Philippines and Senegal) that had been found to be malaria-negative by microscopy. The panel also contained 42 parasite-negative samples from donors with diseases that might be used in the differential diagnoses of malaria or that contained blood factors known to be common in the community or that could result in false-positive reactions in immunochromatographic tests (Table 2). All negative control samples were confirmed free of Plasmodium parasites by PCR amplification.

Whole blood unless indicated. Sera and plasma samples were reconstituted packed cells b Healthy volunteers with no known current illness or blood abnormality

Figure 4a: Origin of phase 2 P. falciparum wild-type (clinical) samples (n=100) 35 30 25 No. of samples 20 15 10 5 0

Figure 4b: Origin of phase 2 P. vivax wild-type (clinical) samples (n=35) 20

15 No. of samples

10

5

0

ed of Repu Ta bli nz c a Af ric C nia an en Re tra pu l bli c Pe ru Ph ilip pin es

dia

Pe ru

ia lom bia

ria

ya

ga

ia

ma

ia

Ke n

ge

mb

mb o

ne

an

mb

iop

Eth

My

Co

Ca

Table 3: Malaria antigen concentrations (ng/mL) in round 5 wild-type, low parasite density (200 parasites/µL) samples pLDH P. falciparum P. vivax

Un it

HRP2 P. falciparum P. falciparum

Ca

Aldolase P. vivax

Mean Median Maximum Minimum Standard deviation

15.5 12.2 43.0 0.2 11.4

16.8 13.2 47.9 1.6 12.6

11.7 7.0 62.5 0.6 13.2

1.5 1.1 7.7 0.0 1.5

Co lo

28

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Eth

Ni

Se

15.0 3.2 3.3

iop

od

8.1 7.0

ia

l

r

5.4. RDT registration Receipt of each shipment of RDTs at the CDC was recorded in a dedicated RDT register. Temperature monitoring devices were offered to manufacturers free of charge to accompany RDT shipments to the CDC. All RDTs were stored at room temperature (≤ 25 °C) immediately, and temperature monitors were labelled with the date of receipt and forwarded for data extraction and analysis, when applicable.

and four RDTs from each lot against a negative sample was performed after RDTs were maintained for 60 days at room temperature (< 25 °C), 35 °C and 45 °C, at 75% humidity. • Ease-of-use assessment: After technicians had become familiar with the test device, they jointly described its blood safety characteristics, the quality of the instructions, the number of timed steps and the total time to a result, using a standard reference guide (24). • RDT anomalies: During testing, technicians regularly reported RDT anomalies according to the list below and guided by Fig. AS2.1. When anomalies were noted frequently, a digital photograph of at least one example was obtained. • red background • incomplete clearing (of blood in the results window) • failure to flow (blood and buffer did not run the length of the strip) • patchy, broken test lines • diffuse lines • thin lines • strip misplaced in cassette • specimen pad not seen in sample window

5.5. Specimen panel registration All panel specimens were assigned unique identification numbers at the collection sites and stored in aliquots of 50 µL at –70 °C until testing. All data pertaining to specimen identification, storage location and characterization are stored in a secure, dedicated database.

5.6. Test phases The evaluation is divided into two phases. Each lot of RDTs is evaluated independently. Lots 1 and 2 of each product were tested alternately against defined sample sets,1 testing of a set of lot 1 of all products was completed, then a set of lot 2 was tested, until both lots of all products had been tested against all panel samples. Phase 1. A screening step is used to allow selection of RDTs that meet the minimal quality requirements. Products from two lots were evaluated against a panel of 20 culturederived P. falciparum samples at high (2000 parasites/µL) and low (200 parasites/µL) parasite density. To progress to the full evaluation (phase 2), a product evaluated in phase 1 must achieve a minimum PDS of 80% against the samples containing 2000 parasites/µL. Phase 2. Products from two lots are evaluated against a panel of diluted clinical blood samples containing wild-type parasites and a parasite-negative panel, evaluated for heat (thermal) stability and assessed for ease of use. • Performance assessment: The mixed parasite-positive and parasite-negative panel comprised 100 P. falciparum, 35 P. vivax at two parasite densities (200 parasites/µL and 2000 (or 5000)2 parasites/µL) and 100 parasite-negative controls. • Heat stability evaluation: Testing of 15 RDTs from each of two lots against a single culture-derived P. falciparum isolate (Nigeria XII strain, P. falciparum HRP2 sequence type B with a typical antigen concentration) at 200 parasites/µL, five RDTs from each lot against P. falciparum Nigeria XII strain at 2000 parasites/µL,

5.7.  Performing rapid tests All RDTs were maintained at room temperature until first use. When applicable, the desiccant was inspected for colour changes, and products were discarded if they were present. RDTs were labelled with sample identification number, dilution and date test was performed. The tests were used according to the manufacturer’s instructions, except that the recommended volume of blood was transferred by micro-pipette from the sample tube, and co-packaged blood transfer devices were not used. The result was recorded by a technician at the minimum specified reading time, and a second technician re-read the result within 30 min for internal monitoring and to obtain information for the manufacturer. Technicians were rotated and blinded to sample type and to each other’s results during phase 2. Annexes 1 and 2 give a descriptive, illustrated summary of the test characteristics and steps and a guide to interpretation of results.

1

A sample set consists of 13 P. falciparum specimens and five P. vivax specimens at 200 parasites/µL and 2000 parasites/µL and 13 malarianegative samples. Four (4%) of the 100 P. falciparum dilution samples sets were at 200 and 5000 parasites/µL and two (6%) of the 35 P. vivax dilution sample sets were at 200 and 5000 parasites/µL.

2

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Methods 29

• ghost test lines

5.8.  Interpreting the results The results of control and test lines were recorded as negative or positive by each technician. Each test was read against a standard colour chart and the band intensity graded as 0 (no visible band), 1, 2, 3 or 4. If the control line was recorded as “0” (no visible band) by either technician, the test was recorded as invalid. Figs 5 and 6 illustrate the testing sequence at low and high parasite density.

Figure 5: Testing procedure and calculation of panel detection score and band intensity for product A against a sample density of 200 parasites/μL The first reading was at the minimum time specified by the manufacturer; the second reading was up to 30 min latera. A sample is considered detected only if all first test readings, from both lots, are positive, i.e. readings a, b, c and d must be positive.

Product A Lot 1 Lot 2

Test 1

Test 2

Test 3

Test 4

a Reading 1 Reading 2

b Reading 1 Reading 2

c Reading 1 Reading 2

d Reading 1 Reading 2

Detected if 4 positive first readings

Based on the positive results of first test reading (2 tests per lot), the mean band intensity score =a+b+c+d/4 (excluding negative results). a Second

reading results are for internal use only

Figure 6: Testing procedure and calculation of panel detection score and band intensity for product A against a sample density of 2000 parasites/μL The first reading was at the minimum time specified by the manufacturer; the second reading was up to 30 min latera. A sample is considered detected only if all first test readings, from both lots, are positive, i.e. readings a and b must be positive.

Product A Lot 1 Lot 2

Test 1

Test 2

a Reading 1 Reading 2

b Reading 1 Reading 2

Detected if 2 positive first readings

Based on positive results of first test reading (2 tests per lot), in each lot, the mean band intensity score =a+b/2 a Second

reading results are for internal use only

30

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

6. Data management Receipt of products was hand-recorded in a RDT register at the CDC as per standard operating procedures. Data associated with specimen collection and characterization were recorded first on hard-copy report forms as per the standard operating procedure at the collection sites (Fig. 2), the Hospital of Tropical Diseases (quantitative ELISA results) and the CDC (PCR results) and then entered directly into Excel followed by importation into a specially developed database. The results of product panel testing and heat stability testing conducted at the CDC were recorded on report forms by each technician individually, as per the standard operating procedure. The results were entered in duplicate and analysed for discrepancies. All source documents and electronic records of the study data are maintained in secure storage until the conclusion of the evaluation, data analysis and publication of the report. Individual product testing reports were sent to manufacturers on 14 March 2014 for a 30-day review period before publication of the final report. Raw data were made available to manufacturers upon request.

7.  Quality assurance Product testing follows standard operating procedures developed during previous testing, which are based on recommendations by expert consultants, with minor modifications by the Steering Committee before round 5 (24). In particular, alternate testing of lots 1 and 2 on discrete sample sets, instead of sequential testing, was introduced, as was a standardized set of descriptions and terms for recording RDT anomalies. Overall, the quality of critical steps was controlled as described below. technician were used for internal monitoring. The summarized results were reviewed in detail, and potential discrepancies were identified and cross-checked against source laboratory report forms. All wild-type parasite samples used in phase 2 were randomized with parasite-negative samples and re-labelled for blinded reading of the RDT results.

7.3.  Quality of WHO specimen bank samples Standard operating procedures were established for the preparation of all specimen bank samples (25). Culture lines of parasites and wild-type samples were selected on the basis of previous evidence and data from specific studies. All diluted parasite samples were stored and transported at –70 °C and were used only once within 8 h of thawing.

7.1.  Quality of malaria RDTs and their use All RDTs were stored in a controlled environment at room temperature (≤ 25 °C). The pouch was opened, and, if applicable, the desiccant was checked for colour change immediately before use. The manufacturer’s instructions were followed, except for use of the blood transfer device provided by the manufacturer: a micropipette was used to ensure the correct blood volume. A temperature monitoring device was offered to manufacturers to be shipped with the RDTs to the testing site (CDC). Logs were analysed for temperatures above or below the manufacturer’s recommended storage conditions.

7.4.  Quality of the product testing site The Division of Parasitic Diseases and Malaria, Center for Global Health, CDC, is the main operating component of the Department of Health and Human Services of the USA, which deals with malaria control and prevention. Laboratories within the Division are accredited by Clinical Laboratory Improvement Amendments and are monitored by an internal quality management system.

7.2.  Quality and objectivity of the RDT reading results The results were read under good lighting by trained technicians tested for visual acuity and doubly entered into the database. Technicians were rotated. The readings of a second

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Methods 31

8. Ethical considerations Each specimen collection site obtained approval from a WHO research ethics review committee and/or a local institutional review board for specimen collection, transport and archiving of blood samples for the purpose of product testing, lot testing and quality assurance.

9. Data analysis 9.1. Measures of parasite detection: panel detection score and positivity rates As shown in Figure 5, a product must return four positive test results at the manufacturers’ recommended minimum reading time (two from lot 1, two from lot 2 at the initial reading time) when tested against a parasite density of 200 parasites/µL to contribute to its PDS. When tested against 2000 or 5000 parasites/µL (Fig. 6), the product must return two positive tests at the manufacturers’ recommended minimum reading time (one from each lot). Thus, the PDS is a measure of inter-test and inter-lot consistency, as well as the ability of the test to detect antigen. The PDS for P. falciparum indicates an RDT result that confirms the presence of P. falciparum when tested against cultured and wild-type P. falciparum samples, while the P. vivax PDS indicates Plasmodium-positive/P. falciparum-negative results when tested with wild-type P. vivax samples. The positivity rate is the percentage of all tests of a particular product that returned a positive result at the manufacturers’ recommended minimum reading time when tested against a P. falciparum or P. vivax sample.

9.2.  False-positive results False-positive results are analysed and reported as two groups: those with incorrect species identification and those that returned a positive result for samples that do not contain Plasmodium spp. Specifically, the false-positive rate is the percentage of all tests of a particular product that returned a positive test result when it should not have, in results obtained at the manufacturer’s recommended minimum reading time.

9.2.1  Incorrect species identification A test is considered to have returned an incorrect species result if a positive P. falciparum test line appears when testing a sample containing non-P. falciparum (P. vivax) parasites. Fig. 7 illustrates the various possibilities for incorrect species identification in combination tests. For example, if P. falciparum samples result in only a visible pan-specific (or non-P. falciparum -specific) test line in combination

Figure 7: Classification of incorrect species identification with combination malaria RDTs Pf/pan combination tests Panel sample Pf Pv False-positive (Pf) False-positive (Pf) Pf + / Pan Pf + / Pan + Pf - / Pan + False-positive (non-Pf) Pf - / Pan Negative Negative

Pf/Pv combination tests Panel Pf Pv False-positive (Pf) False-positive (Pf) Pf + / Pv Pf + / Pv + Pf - / Pv + False-positive (non-Pf) Pf - / Pv Negative Negative

32

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

tests, the result is considered to be a false-positive for non-P. falciparum parasites.

9.5.  Invalid tests Invalid tests are those deemed invalid during testing of both lots, with samples at 200 parasites/µL and 2000 (or 5000) parasites/µL.

9.2.2  False-positive results for Plasmodiumnegative samples Any positive reading of samples with no Plasmodium parasites is considered a false positive. In phase 2, parasite-negative samples are clean-negative samples and samples containing other infectious agents (e.g. dengue, leishmania, Chagas) and immunological factors (e.g. rheumatoid factor, anti-nuclear antibodies, anti-mouse antibodies) (Table 2).

9.6.  Heat (thermal) stability The results of heat stability testing are reported as the number of positive tests against one cultured P. falciparum parasite sample at 200 and 2000 parasites/µL based on the first reading of two lots at each parasite density (maximum score, 30 against 200 parasites/µL samples and 10 against 2000 parasites/µL samples)2 and mean band intensity (for positive tests only based on the first reading) after the lots were stored at room temperature (< 25 °C) and at 35 °C and 45 °C for 2 months.

9.3. Band intensity All positive test results were recorded with their band intensity against a standard reference chart, matched closely to line colour. On the basis of the results of the first reader, the distribution of band intensity results is presented as the mean band intensity of positive results. In addition, the intensity was expressed for each possible result (0, 1, 2, 3 or 4) as the percentage recorded at that level.1

9.4. Lot agreement Agreement between test lots is calculated from the number of samples that return a positive result on both RDTs tested in that lot against parasite-positive samples at 200 parasites/µL, and on the single RDT from each lot tested against samples at 2000 (or 5000) parasites/µL. High inter-lot agreement indicates consistency in detecting malaria parasites. When one test is invalid and the other positive, positive agreement is recorded. Fig. 8 shows sample calculations for lot agreement.

1

A standard intensity comparison chart is used, which allows matching to the closest of four common colour variants of labelled antibodies used in RDTs, each at four levels of intensity.

2

Fifteen tests per lot against 200 parasites/µL samples and 5 tests per lot against 2000 parasites/µL samples. Invalid results were excluded from analysis.

Figure 8: Explanation of lot agreement calculation Test results (1= positive, 0 = negative) Lot 1 Test 1 reader 1 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 1 1 0 0 1 Test 2 reader 1 1 0 1 0 1 Test 1 reader 1 1 0 0 0 1 Lot 2 Test 2 reader 1 1 0 1 0 1 (a) Lot 1 tests 1 Disagree Disagree 0 1 Derived values (1= both positive, 0 = both negative) (b) Lot 2 tests 1 0 Disagree 0 1 (d) Comparison of lot results 1 Can’t compare Can’t compare 0 1 (f) Contribution to overall 1 0 0 0 1

PDS = sum (f) / number of samples = 2/5 = 40 Lot 1 PDS = sum (a) / number of samples = 2 / 5 = 40 Lot 2 PDS = sum (b) / number of samples = 2 / 5 = 40 Positivity = number of positive results / total number of tests = 11 / 20 = 55% Agreement between tests = (count number of 0 and 1s in (a) and (b)) / (number of samples x 2 lots) = 7 / 10 = 70% Agreement between lots = (count number of 0 and 1s in (d)) / (number of samples -­ ‐ number of “can’t compare” in (d)) = 3 /3 = 100% Note: reader 1 = Technician 1 in raw data files

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 33

10. RELATION BETWEEN PARASITE DENSITY AND ANTIGEN CONCENTRATION Malaria RDTs detect parasite-derived antigen. The relation of the concentration of antigen available from the blood sample (after lysis of red cells and parasites) to the peripheral parasite density varies widely because of a series of host and parasite factors (Box 4). In establishing panels for the product testing programme that reflect possible variation in antigen concentration for parasitaemia of 200 parasites/µL, a large number (> 300) of wild-type parasite samples from clinical cases in different geographical areas were analysed by quantitative ELISA for HRP2, pLDH and aldolase. Only samples with antigen values within the 90th percentile for HRP, pLDH and aldolase were selected for the performance panels. Furthermore, the distribution of antigen levels for HRP2, pLDH and aldolase was compared with that in previous rounds to ensure consistency. No statistically significant differences in median antigen levels between the panels for rounds 1–5 were detected for any of the three antigens (p > 0.1, Kruskal-Wallis test). Therefore, the panels used for the product testing rounds can be considered comparable (Annex S1).

Box 4. Explanations for variable antigen concentrations in samples with the same parasite density • variation in antigen expression among isolates • different durations of infections (accumulating antigens) • different parasite growth stages at the time of collection (expressing different levels of antigens) • presence of circulating HRP2 from previous cycles of growth • HRP2 produced by parasites sequestered in the host’s vascular tissues that cannot be accounted for in the estimate of parasite density on the blood slide (29)

11. Laboratory versus field-based malaria RDT evaluations Despite the strengths of the product testing programme, the evaluations are not completely analogous to field testing of malaria RDTs. In order to compose a panel that could be used to evaluate RDTs reproducibly, blood samples must be diluted, frozen and stored below −70 °C; however, blood that has undergone freezing and thawing is lysed and may not have exactly the same characteristics as fresh blood. Another difference from field evaluation is use of a micro-pipette to place blood in the RDT device rather than the blood transfer device provided by the manufacturer. This is necessary because blood is collected from a cryo-tube rather than a finger-prick, and the blood transfer devices provided with the different products vary (30). This technique also ensures the consistency of testing by reducing the likelihood of operator error. As all samples in the panel used for the evaluation are prepared from parasites that express HRP2, the results will not be predictive of field trial results of parasite populations with significant levels of HRP2 deletion (10, 11). In addition, the population frequency of blood immunological factors or infectious diseases, which can result in false-positive results, may vary. Therefore, the sensitivity and specificity of an RDT in the field depends on the epidemiological situation. The evaluation reported here does not predict sensitivity or specificity in a given field situation but the rates of detection of target antigens and falsepositive results of RDTs against a standardized panel in a controlled, repeatable manner. As the panel is meant to be a close approximation of field samples, the detection rates of different products will be reflected in similar differences in the field. The panel is designed to include a large number of samples that are close to the limit of detection of RDTs (200 parasites/µL) and is therefore likely to discriminate between them more clearly than a field trial. It follows that in settings where parasite density is very high, no differences in the PDS and positivity rates of tests or much smaller differences will be observed compared to those reported against the WHO evaluation panel. Furthermore, where the parasite density is very low, the detection rates may be lower than those reported here. Field trials have a place in product selection, particularly in determining which of a short-list of products is most appropriate for the technicians and situation of its intended

34

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

use in a programme (e.g. ease-of-use characteristics). Such trials should have carefully defined objectives and procedures designed to achieve them. Trials to determine the probable field sensitivity and specificity of a product also have a place but require large samples and populations with low parasite densities if significant differences are to be found

between well-performing products; they must also be closely controlled and are therefore expensive. Such trials do not allow comparison of a large number of products. WHO has published recommendations for good practice in malaria field trials (31), which should be followed to improve the reproducibility and quality of the results.

12. Results 12.1. Summary Round 5 of WHO malaria RDT product testing provided results for 42 products evaluated against P. falciparum culture samples, and all the products proceeded to evaluation against wild-type samples collected from parasitaemic patients on three continents and a large panel of parasite-negative samples. Heat stability was assessed at the temperatures commonly encountered in malaria-endemic countries. Thirteen research institutes were engaged in either sample collection or sample characterization to establish the evaluation panels. Between January 2013 and December 2013, approximately 58 400 RDTs were tested at the CDC. The main results are presented in Tables 4 and 5, which group the RDTs by the species they are designed to detect, i.e. P. falciparum only, P. falciparum and non-falciparum species, P. falciparum and P. vivax, P. vivax, ovale and malariae, pan only or all malaria species without discrimination. Note that only tests against P. falciparum and P. vivax samples were evaluated, and the evaluation does not therefore indicate whether a product intended to detect other species can do so. PDS at both high and low parasite concentrations are presented, as are false-positive rates and the percentage of invalid test results. Tests in each category are listed alphabetically, but the results are colour-coded according to WHO-recommended RDT procurement selection criteria (Box 3). When choosing an appropriate product, it is important also to review its thermal stability (Table 6) in the context of the expected conditions of transport and storage in the field. The results of the evaluation are listed below. • The overall range of results against wild-type and negative samples, including PDS, positivity rate, false-positive rate and heat stability, were similar to those reported in rounds 1–4 (3–6). The median PDS for P. falciparum was slightly lower at low parasite densities than in the previous round (89.3% vs 85%), and no products scored a PDS of 100% in round 5. The PDS for P. vivax at low densities has improved consistently since round 1 (median, 30%), the results for rounds 2, 3, 4 and 5 being 75.0%, 51.4%, 61.8% and 65.7% respectively. The median false-positive rate remained consistently at about 1%; however, the range of false-positive rates increased significantly with each round of testing (upper limit in rounds 1–5: 28%, 37%, 44%, 99.1% and 81.3%). Some products with high false-positive rates also had high PDS, underscoring the importance of reviewing all aspects of product performance. • A number of RDTs consistently detected malaria at a low parasite density (200 parasites/µL), had low false-positive rates, were stable at tropical temperatures, are relatively easy to use and can detect P. falciparum, P. vivax or both infections, increasing the number of available wellperforming tests from that in rounds 1–4.

• P. falciparum tests targeting the HRP2 antigen had the highest PDS for P. falciparum, and the two products with the poorest performance at 200 parasites/µL targeted P. falciparum -specific pLDH antigen. Two resubmitted pan-only tests maintained good PDS for P. falciparum at low parasite density. • Several combination tests achieved PDS at the upper end of the range for both P. falciparum and P. vivax. (Fig. S3). • The test performance of some products varied between lots. • Most combination tests in which HRP2 is used for detection of Pf return positive results predominantly only on the HRP2 band at lower densities of Pf. This reinforces manufacturers instructions to classify Pf infections as either HRP2 test line-positive alone or in combination with the pan-pLDH line. Tables 4 and 5 summarize the performance of malaria RDTs against cultured P. falciparum parasites, blood containing wild-type P. falciparum and P. vivax parasites and Plasmodium spp.-negative samples. Detailed phase-1 and phase-2 results of product testing are given in annexes 3 and 4, respectively. The data are shown graphically in Figs 9–27.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 35

• The performance of products varied widely at low parasite density (200 parasites/µL), but most showed a high detection rate for P. falciparum and P. vivax at 2000 (or 5000) parasites/µL.

36

Table 4: Summary phase-1 performance of 42 malaria RDTs against 20 cultured P. falciparum lines at low (200) and high (2000) parasite density (parasites/μL) Panel detection scorea (n=20) Manufacturer 200 parasites/μL 95.0 85.0 100.0 100.0 100.0 95.0 95.0 100.0 100.0 85.0 95.0 0.0 90.0 95.0 100.0 100.0 100.0 95.0 95.0 90.0 90.0 85.0 95.0 60.0 100.0 100.0 5.0 100.0 100.0 100.0 100.0 95.0 100.0 90.0 100.0 90.0 95.0 95.0 100.0 100.0 100.0 100.0 95.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 0.0 (78) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.0 0.0 0.0 (77) 3.8 0.0 0.0 (79) 0.0 0.0 0.0 0.0 0.0 11.3 1.3 (78) 13.8 53.8 100.0 100.0 100.0 100.0 100.0 100.0 100.0 (19) 100.0 100.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 (39) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 97.5 0.0 95.0 35.0

False-positive non-Pf infectionb (%) 200 parasites/μL (n=80) 2000 parasites/μL (n=40)

Product 2000 parasites/μL

Catalogue number

Invalid rate (%) (n=160)

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd

0.0 0.0 0.0 0.0 0.0 0.6 1.3 0.0 0.0 2.5 0.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0 0.0 0.6 0.0 0.0 0.0 0.0 0.6 0.0 1.3 0.0 0.0

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) ICT INTERNATIONAL Artron Laboratories Inc. Humasis Co., Ltd. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd.

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan Antigen Test Malaria Pf/Pan One Step Rapid Test Malaria pf-LDH/PAN-LDH Antigen Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette)

I16FRC MALAG100 20-0146-01 ML03 A03-18-322 AMAL-7025 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

AM4650-K GM006 MAT-PF/PAN-50 523352

Table 4 (continued) Panel detection scorea (n=20) Manufacturer 200 parasites/μL 95.0 95.0 65.0 95.0 100.0 100.0 NA NA NA NA 100.0 100.0 1.3 2.5 0.0 2.5

False-positive non-Pf infectionb (%) 200 parasites/μL (n=80) 2000 parasites/μL (n=40)

Product 2000 parasites/μL Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc.

Catalogue number

Invalid rate (%) (n=160) 0.0 0.0 0.0 0.0

ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

55IC402-50 W056-C

IR013025 G0111

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a A sample is considered detected only if all RDTs from both lots read by the first technician, at minimum specified reading time, are positive b Pan or Pv line only positive indicates a false-positive non P. falciparum infection

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results

37

38

Table 5: Summary phase-2 performance of 42 malaria RDTs against wild-type (clinical) P. falciparum and P. vivax samples at low (200) and high (2000a) parasite density (parasites/μL) and Plasmodium spp. negative samples Panel detection scoreb False-positive rates (%) 200 parasites/μL Pv samples Falsepositive Pf infectiond (n=140) 3.6 0.7 0.0 2.1 0.7 3.6 11.4 0.0 2.1 0.3 0 (139) 0.3 (389) 6.7 (134) 1.5 0.7 3.5 0.0 0.8 0.7 0.5 0.7 1.5 0.7 0.3 12.1 0.3 2.9 100.0 100.0 54.3 17.1 91.4 40.0 2.9 91.4 8.6 28.6 90.0 86.0 92.0 74.0 65.7 88.6 37.1 40.0 100.0 96.0 100.0 98.0 95.0 100.0 97.0 100.0 100.0 100.0 100.0 94.0 100.0 100.0 97.1 88.6 97.1 94.3 97.1 100.0 45.7 40.0 94.3 97.1 97.1 88.6 0.3 0.3 0.0 0.0 0.5 (396) 0.3 0.0 (398) 0.0 (398) 22.5 0.0 0.5 (399) 0.0 (399) 0.5 2.0 0.7 (139) 0.0 0.0 13.6 0.0 (138) 4.3 4.3 0.7 (138) 47.9 38.6 9.3 0.0 0.7 2.9 NA NA NA NA NA NA NA NA NA 0.0 (199) 0.0 (197) 0.5 0.0 0.5 (198) 2.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 (199) 0.5 0.0 (199) 0.0 (199) 1.5 0.0 0.0 0.0 0.0 (199) 0.5

200 parasites/μL Manufacturer Pf samples (n=100) Pv samples (n=35) Pf samples (n=100) Pv samples (n=35) Pf samples Falsepositive non-Pf infectionc (n=400) NA NA NA NA NA NA NA NA NA

2000 parasites/μL

Product

Catalogue number

Total falsepositive ratese (%) Clean2000 parasites/μL Invalid negative rate (%) Pf samples Pv samples samples (n=1214) FalseFalse-positive Falsepositive positive Pf Plasmodium non-Pf d spp. infection infectionc infection (n=236) (n=70) (n=200) 5.7 0.0 0.0 1.4 0.0 4.3 12.9 2.9 1.4 0.0 1.4 0.0 0.0 (69) 0.0 0.0 0.0 7.1 1.5 (67) 0.0 0.0 0.0 7.1 1.4 2.9 0.0 0.0 (69) 35.7 40.0 4.3 1.4 1.4 5.7 7.7 (233) 0.0 0.9 0.9 (235) 0.4 5.1 (234) 10.6 (235) 0.0 5.1 (235) 7.3 (234) 8.7 (231) 0.4 0.0 4.7 0.4 0.4 12.7 2.1 1.3 (235) 0.0 0.0 (235) 10.6 0.9 (235) 3.0 0.9 0.4 (232) 81.3 (235) 39.4 15.3 1.3 4.2 14.0 0.4 0.0 0.0 0.3 0.0 0.2 0.7 0.0 0.1 0.4 2.1 0.0 0.2 0.2 0.0 0.0 0.0 0.2 0.3 0.0 0.2 0.1 0.7 0.0 0.2 1.0 0.1 0.0 0.1 0.1 0.1 0.0

Pf only InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 66.0 88.0 30.0 84.0 77.0 77.0 90.0 89.0 90.0 83.0 85.0 84.0 67.0 90.0 93.0 61.0 89.0 41.0 95.0 74.3 80.0 37.1 60.0 94.3 100.0 77.1 77.1 94.3 60.0 82.9 92.0 100.0 94.0 100.0 99.0 94.0 100.0 99.0 100.0 97.1 97.1 97.1 100.0 97.1 100.0 100.0 85.7 97.1 89.0 91.0 88.0 95.0 72.0 79.0 95.0 84.0 NA NA NA NA NA NA NA NA 99.0 100.0 100.0 100.0 93.0 91.8 (98) 99.0 100.0 NA NA NA NA NA NA NA NA 53.0 NA 93.0 NA

ITP11002TC1/ TC40 Advantage P.f Malaria Card IR016025 CareStart™ Malaria HRP2 (Pf) G0141 diagnosticks- Malaria (Pf)Cassette WB KMFC6001 First Response® Malaria Ag P. falciparum (HRP2) Card Test I13FRC IMMUNOQUICK® MALARIA falciparum 0502_K25 KHB® Malaria Ag P.f Rapid Test KH-R-06-20 SD BIOLINE Malaria Antigen Pf 05FK50/05FK53 Vision Malaria Pf VB01 Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25 Advanced Quality™ Rapid Malaria Test (Pf/Pan) ITP11005 Advantage Mal Card IR221025 Advantage Malaria Pan + Pf Card IR231025 BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) C30RHA25 BioTracer™ Malaria Pf/PAN Rapid Card 17012 CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo G0131 Clearview® Malaria Dual VB20 DIAQUICK Malaria P.f/Pan Cassette Z11200CE LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd.

Advanced Quality™ One Step Malaria Pf Test

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria Pf/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test

I16FRC

NG-Test MALARIA Pf/Pan (pLDH)

OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette

MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NGMAL-W23-001 R0113C 50310025 IMPN-C52

Table 5 (continued) Panel detection scoreb False-positive rates (%) 200 parasites/μL Pv samples Falsepositive Pf infectiond (n=140) 0.7 0.7 (139) 0.0 6.5 (138) 5.8 (139) 53.6 5.7 2.9 NA NA 0.5 1.5 NA NA 85.5 3.6 (195) 87.4 (199) 9.0 0.5 0.5 (199) 1.4 0.0 (69) 0.0 2.9 4.3 (69) 34.3 2.9 2.9 NA NA Performance measure Panel detection score for Pf and Pv 200/µL samples

200 parasites/μL Manufacturer Pf samples (n=100) 94.0 86.0 81.0 85.0 84.0 92.0 63.0 87.0 77.0 84.0 100.0 88.6 98.0 99.0 100.0 97.1 NA NA 28.6 98.0 97.1 1.5 (399) 37.1 91.0 85.7 2.0 (399) 34.3 74.3 62.9 100.0 99.0 97.0 100.0 100.0 100.0 94.3 100.0 100.0 16.5 1.5 (391) 27.3 (399) 21.5 91.4 5.7 99.0 97.0 97.1 94.3 0.8 0.0

2000 parasites/μL Pf samples (n=100) Pv samples (n=35) Pf samples Falsepositive non-Pf infectionc (n=400)

Product Pv samples (n=35)

Catalogue number

Total falsepositive ratese (%) Clean2000 parasites/μL Invalid negative rate (%) Pf samples Pv samples samples (n=1214) FalseFalse-positive Falsepositive positive Pf Plasmodium non-Pf d spp. infection infectionc infection (n=236) (n=70) (n=200) 0.0 1.3 (235) 0.4 (235) 0.9 (232) 3.0 (232) 77.1 6.4 2.1 0.4 0.0 0.0 0.3 0.2 2.5 0.7 0.0 0.1 0.1 0.0 0.0 Recommended WHO procurement criteria ≥ 75% < 10%

05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc.

AM4650-K GM006 MAT-PF/PAN-50 523352

55IC402-50

SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™ Malaria pLDH (PAN)

W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a 4 (4%) of the 100 P. falciparum dilution sample sets had 200 and 5000 parasites/μL and 2 (6%) of the 35 P. vivax dilution sample sets had 200 and 5000 parasites/μL b A sample is considered detected only if all RDTs from both lots read by the first technician, at minimum specified reading time, are positive c For combination tests, pan or Pv line, only, positive indicates a false-positive non P. falciparum infection d Pf line positive indicates a false-positive P. falciparum infection e The total number of times a positive result for malaria was generated when it should not have been

False-positive rates against clean-negatives Invalid rate < 5% of tests conducted

Results

39

12.2.  Phase 1: P. falciparum culture panel All the tests consistently detected ≥ 95% of cultured P. falciparum parasites at high parasite density (2000 or 5000 parasites/µL); however, the PDS was highly variable (0–100%) at low parasite density (200 parasites/µL). At low parasite density, the products with the highest PDS targeted HRP2 (Fig. 9).

Figure 9: Phase-1 P. falciparum panel detection score of malaria RDTs at low (200) and high (2000) parasite density (parasites/μL) according to target antigen type (HRP2 or pLDH)

Panel detection scorea

200 (HRP2) 200 (pLDH) 2000 (HRP2) 2000 (pLDH)

a A

sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive.

40

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

12.3.  Phase 2: Wild-type P. falciparum and P. vivax and Plasmodium spp.-negative samples 12.3.1  P. falciparum detection All 42 products in round 5 were designed to detect P. falciparum. As in phase 1, most of the tests (34; 81%) had a PDS ≥ 95% of P. falciparum samples at high parasite densities. Seven of the nine products specific for P. falciparum alone achieved a PDS ≥ 75% against samples with low parasite density (Fig. 10).

Figure 10: Phase-2 P. falciparum panel detection score of malaria RDTs at low (200) and high (2000a) parasite density (parasites/μL) according to target antigen type (HRP2 or pLDH)b

Panel detection scorec

200 (HRP2) 200 (pLDH) 2000 (HRP2) 2000 (pLDH)

(4%) of the 100 P. falciparum dilution samples sets had 200 and 5000 parasites/μL and 2 (6%) of the 35 P. vivax dilution sample sets had 200 and 5000 parasites/μL. b Phase-2 evaluation panel consisted of 100 clinical blood samples containing wild-type P. falciparum. RDTs performed = 2 tests x 2 lots at 200 p/μL and 1 test x 2 lots at 2000 p/μL. c A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive.

a 4

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 41

12.3.2  P. vivax detection Fig. 11 shows that 31 of 33 (94%) products designed to detect P. vivax consistently detected ≥ 75% at high parasite density (2000 or 5000 parasites/µL), and 14 (42%) achieved the same threshold of PDS against samples with 200 parasite/µL. The overall detection rate in low parasite density wild-type P. vivax samples was lower than that for P. falciparum. At a low parasite density (200 parasite/µL), only 8 products had a PDS ≥ 90%, and 19 had a PDS < 75% (Table 5).

12.3.3 Combined detection of P. falciparum and P. vivax Of the 33 pan-specific and combination tests, 13 (39%) had a PDS ≥ 75% for both P. falciparum and P. vivax at a low parasite density (200 parasites/µL) (Table 5). These included the two pan-specific only tests. Several performed well at a high parasite density.

Figure 11: Phase-2 P. vivax panel detection score of malaria RDTs at low (200) and high (2000a) parasite density (parasites/μL) according to target antigen type (aldolase, pLDH)b

Panel detection scorec

200 (pLDH) 2000 (pLDH) 200 (aldolase) 2000 (aldolase)

a 2

(6%) of the 35 P. vivax dilution sample sets had 200 and 5000 parasites/μL. evaluation panel consisted of 35 clinical blood samples containing wild-type P. vivax; RDTs performed = 2 tests x 2 lots at 200 p/μL and 1 test x 2 lots at 2000 p/μL. c A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive. b Phase-2

42

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

12.3.4  P. falciparum and P. vivax positivity rate The positivity rate was measured in addition to the PDS, to measure the number of times a test returned a positive result. As expected, the positivity rates were higher than the PDS but mirrored the PDS against wild-type P. falciparum and P. vivax samples (Figs 12 and 13).

12.3.5 Band intensity Although RDTs do not provide quantitative results, the technicians graded positive results according to a standard colour chart and calculated the mean band intensity for positive results (Annex 4, tables A4.2 and A4.3). A positive correlation was found between the PDS and band intensity (Spearman rank correlation, r = 0.67, p  < 0.001 for the P. falciparum panel and r  = 0.66, p  < 0.001 for the P. vivax panel).

Figure 12: Phase-2 P. falciparum panel detection score and positivity rate at 200 parasites/μL a

Panel detection scoreb/Positivity ratec (%)

Positivity rate (HRP2) Positivity rate (pLDH) Panel detection score (HRP2) Panel detection score (pLDH)

a Phase-2 evaluation

panel consisted of 100 clinical blood samples containing wild-type P. falciparum. RDTs performed = 2 tests x 2 lots at 200 p/μL and 1 test x 2 lots at 2000 p/μL. b A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive. c The total number of times a test returned a positive result divided by the total number of times it should have (x 100).

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 43

Of the combination RDT products containing a pan test band that gave a positive indication for P. falciparum against low-density P. falciparum samples, 44.1% (3850/8721) gave positive results on both the P. falciparum and pan test bands, and 55.9% (4871/8821) were positive only on the P. falciparum test band. Of HRP2-detecting products, 41.9% (3454/8247) of those that detected P. falciparum showed both the P. falciparum (HRP2) and the pan test band (pan-pLDH) as positive, while 58.1% (4793/8247) were positive only on the P. falciparum HRP2 test band. The values were 83.5% (396/474) and 16.5% (78/474) for the non-HRP2-based products.

When the pan test band in the combination products was positive, the mean intensity of the band was 1.07 (standard deviation, 0.23), which was significantly lower than the corresponding mean P. falciparum test band intensity (mean, 2.04; standard deviation, 0.42), when tested against the lower-density P. falciparum parasite panel (paired t test, p  < 0.001). There was no correlation between the mean intensity of the pan test band and the P. falciparum test band (r = –0.049, p  = 0.805).

Figure 13: Phase-2 P. vivax panel detection score and positivity rate at 200 parasites/μL a

Panel detection scoreb/Positivity ratec (%)

Positivity rate (pDLH) Positivity rate (aldolase) Panel detection score (pLDH) Panel detection score (aldolase)

a Phase-2

evaluation panel consisted of 35 clinical blood samples containing wild-type P. vivax; . RDTs performed = 2 tests x 2 lots at 200 p/μL and 1 test x 2 lots at 2000 p/μL. b A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive. c The total number of times a test returned a positive result divided by the total number of times it should have (x 100).

44

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

12.3.6  False-positive rates Eight (19%) tests had false-positive rates > 10% on 59 clean-negative samples on any test line (Figs 14 and 15). The same eight tests also had high false-positive rates for samples containing other pathogens and immunological abnormalities.

Only one test with a low false-positive rate (< 10%) on cleannegative samples returned high false-positive rates (range, 8.3–100%) against samples from patients with other blood pathogens. Several tests with low false-positive rates (< 10%) on clean-negative samples had high false-positive rates against samples with immunological blood abnormalities,

Figure 14: Phase-2 P. falciparum (P. falciparum test line) false-positive rate against clean-negative samplesa

False-positive rate (%)

pLDH HRP2

a Phase-2

evaluation panel consisted of 100 Plasmodium spp.-negative samples, of which 59 were clean negatives from healthy volunteers with no known current illness or blood abnormality.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 45

particularly those containing rheumatoid factor and human anti-mouse antibody. There were, however, only 13 samples containing non-Plasmodium infectious agents and 29 samples containing immunological factors. For detailed information on the blood abnormalities and pathogens that generated false-positive results in specific products, see Annex 4 (tables A4.8 and A4.9).

Importantly, there was no clear trend of higher false-positive rates in tests with higher PDS, indicating no clear trade-off between the sensitivity and specificity of the tests at these detection thresholds (Figs 16 and 17).

Figure 15: Phase-2 Plasmodium spp. (pan or P. vivax/Pvom test line) false-positive rate against clean-negativesa pLDH Aldolase

a Phase-2

evaluation panel consisted of 100 Plasmodium spp.-negative samples, of which 59 were clean negatives from healthy volunteers with no known current illness or blood abnormality.

46

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

False-positive rate (%)

Figure 16: Phase-2 P. falciparum false-positive ratea versus P. falciparum panel detection score (PDS)b at low (200) parasite density (parasites/μL)

False-positive rate (%)

P. falciparum PDS at 200 parasites/µL a False-positive

rate is for clean-negatives only. b A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive.

Figure 17: Phase-2 P. vivax false-positive ratea versus P. vivax panel detection scoreb at low (200) parasite density (parasites/μL) pLDH Aldolase

False-positive rate (%)

P. vivax PDS at 200 parasites/μL a False-positive

rate is for clean-negatives only. b A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 47

12.4.  Performance of products under the compulsory resubmission requirement With the new requirement for compulsory resubmission of products within 5 years of testing and the limit of one voluntary submission per manufacturer, over half (55%) of the products in round 5 had been evaluated previously. For 17 (74%) of the resubmissions, this was the second testing, and six had been tested more than twice. Figs 18 and 19 show the performance in first and second testing of products against wild-type P. falciparum and P. vivax at 200 parasites/µL and clean-negative samples that had been resubmitted compulsorily and voluntarily.

For the 10 products that had not been tested since round 1 (compulsory resubmissions), a significant correlation was found between the PDS in rounds 1 and 5 for P. falciparum at low density (Spearman rank correlation, r  = 0.92, p  < 0.001). The median change in PDS between the two rounds was –7.6% (range, –32.0 to 4.8%), which was significantly different from zero (Wilcoxon signed rank test, p  = 0.013). As only four of the products detect non-falciparum parasites, no statistical analysis was conducted for detection of the P. vivax parasite panel. The change in performance between rounds followed a different pattern for the 13 products that were voluntarily resubmitted for testing. No significant correlation was found in the PDS for P. falciparum at lower parasite density in

Figure 18: Phase-2 P. falciparum panel detection scorea at low (200) parasite density (parasites/μL) during initial and subsequent testing of compulsorily and voluntarily resubmitted malaria RDTs False-positive Plasmodium spp. rate on clean-negative samplesb (%) Compulsory retest Voluntary retest Panel detection score (prior submission) Panel detection score (round 5) False-positive (prior submission) False-positive (round 5) a Panel b Clean-negative,

Panel detection scorea 48 Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

detection score: A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive. blood samples from healthy volunteers with no known current illness or blood abnormality.

consecutive submissions (Spearman rank correlation, r = 0.32, p  = 0.29). The median change in detection of P. falciparum was 6.5% (range, –10.0 to 42.0%), which was significantly different from zero (Wilcoxon signed rank test, p  = 0.033). Most of these products (11/13) detected P. vivax, and detection of this parasite improved overall (median change, 10.6%; range, –20.0 to 100.0%), although the change was not statistically significant (Wilcoxon signed rank test, p = 0.075). No correlation was found in the PDS against P. vivax at a lower parasite density of consecutive submissions (Spearman rank correlation, r  = –0.032, p  = 0.925).

Importantly, there was little change in the false-positive rates of the resubmitted products, with four exceptions. The high false-positive rates (> 25%) of two voluntarily resubmitted products in prior testing were reduced to < 5% in round 5, while their ability to detect P. vivax was improved, with minimal loss of ability to detect P. falciparum. The opposite result was found for another two voluntarily submitted products with low false-positive rates (< 2%) in prior testing, which returned exceptionally high false-positive rates (> 75%) in round 5. These changes were associated with a higher PDS for P. falciparum at a lower density and mixed results for P. vivax detection.

Figure 19: Phase-2 P. vivax panel detection scorea at low (200) parasite density (parasites/μL) during initial and subsequent testing of compulsorily and voluntarily resubmitted malaria RDTs False-positive Plasmodium spp. rate on clean-negative samplesb (%)

Panel detection scorea

Compulsory retest

Voluntary retest Panel detection score (prior submission) Panel detection score (round 5) False-positive (prior submission) False-positive (round 5)

a Panel

b Clean-negative,

detection score: A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive. blood samples from healthy volunteers with no known current illness or blood abnormality.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 49

13.  Heat stability A single P. falciparum culture sample (from the same lot as tested in rounds 3 and 4) was used as the reference sample to test heat stability. This was necessary because establishment of a continuous in vitro culture system for P. vivax is very difficult, largely because of the selective invasion of reticulocytes by the parasite. Therefore, it is not possible to provide stability data on test lines that detect only non-P. falciparum parasites. Such data, and confirmatory data on the stability of recent production lots of all tests, should be obtained from manufacturers and through the WHO-FIND lot-testing programme during product selection for procurement of RDTs. All the P. falciparum -only RDT test lines were heat-stable. Thus, they detected a cultured P. falciparum sample the same number of times when stored at room temperature (< 25 °C) or at 35 °C or 45 °C (75% humidity) for 2 months (Fig. 20). Overall, the products were more stable against samples with high (2000 parasites/µL) rather than low (200 parasites/µL) parasite density (Figs 21, 23, 25 and 27 and Figs 20, 22, 24 and 26, respectively), as little deterioration will not be apparent at high parasite density. The stability of pan-pLDH-detecting test lines was much poorer than that of HRP2-detecting test lines, at both high and low parasite density. Most combination test products have pan lines that poorly detect low-density P. falciparum samples at baseline. Furthermore, tests with a baseline positivity < 100% showed unpredictable variation in positivity rates on subsequent testing, indicating that they were on the borderline of visibility. One of the two combination tests with good baseline pan line reactivity to the low-density sample showed a marked reduction in performance after 2 months at 35 °C or 45 °C. Some combination P. falciparum test lines were stable at 35 °C but lost the ability to detect antigen after incubation at 45 °C. As reported previously, a few products showed better performance after incubation (Figs 21, 22, 24, 26, 27). The results of heat and thermal stability testing are summarized in Table 6, and detailed results are presented in Annex 4 (tables A4.11–A4.13a) and in Figs 20–27, which show the results for the two lots combined (maximum score, 30; 15 tests per lot against 200 parasites/µL; maximum score, 10; 5 tests per lot against 2000 parasites/µL).

50

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Table 6: Heat stability testing results for 42 malaria RDTs on a cultured P. falciparum sample at low (200) and high (2000) parasite density (parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °Ca Positive test results for P. falciparum (Pf line) Positive test results for P. falciparum (pan line) 200 parasites/μL Baseline Number of tests positive (max. 30) Lots 1 and 2 combined NA NA NA NA NA NA NA NA NA 1.0 0.0 0.0 24.0 0.0 0.0 28.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 3.0 0.0 4.0 0.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 0.0 0.0 3.0 0.0 3.0 0.0 0.0 0.0 0.0 1.0 0.0 28.0 0.0 2.0 0.0 1.0 3.0 0.0 0.0 28.0 16.0 0.0 26.0 3.0 0.0 0.0 0.0 0.0 8.0 0.0 20.0 16.0 1.0 0.0 1.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 30.0 0.0 0.0 1.0 4.0 10.0 10.0 10.0 10.0 9.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 9.0 10.0 10.0 10.0 10.0 10.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 NA NA 10.0 8.0 8.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0

Positive test results for P. falciparum (Pf line) 2000 parasites/μL Baseline Number of tests positive (max. 10) Lots 1 and 2 combined 35 °C 45 °C Baseline 35 °C

Positive test results for P. falciparum (pan line) 2000 parasites/μL 45 °C Number of tests positive (max. 10) Lots 1 and 2 combined NA NA NA NA NA NA NA NA NA 7.0 7.0 10.0 10.0 9.0 10.0 10.0 9.0 10.0 10.0 10.0 5.0 0.0 10.0 9.0 10.0 10.0 6.0 10.0 10.0 9.0 10.0 NA NA NA NA NA NA NA NA NA 9.0 10.0 10.0 10.0 10.0 10.0 10.0 9.0 10.0 10.0 10.0 10.0 0.0 10.0 9.0 10.0 9.0 10.0 10.0 10.0 10.0 10.0 NA NA NA NA NA NA NA NA NA 3.0 8.0 10.0 10.0 10.0 10.0 10.0 10.0 8.0 10.0 10.0 1.0 0.0 10.0 9.0 9.0 10.0 10.0 10.0 10.0 10.0 10.0

Product Baseline Number of tests positive (max. 30) Lots 1 and 2 combined InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 25.0 26.0 0.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 3.0 30.0 14.0 30.0 30.0 30.0 30.0 30.0 30.0 2.0 30.0 17.0 30.0 30.0 28.0 13.0 30.0 30.0 0.0 29.0 20.0 30.0 30.0 30.0 22.0 29.0 0.0 30.0 30.0 29.0 30.0 30.0 30.0 3.0 30.0 0.0 30.0 29.0 27.0 29.0 30.0 29.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 30.0 28.0 29.0 27.0

Catalogue number

Manufacturer 35 °C 45 °C 35 °C 45 °C

200 parasites/μL

Pf only

Advanced Quality™ One Step Malaria Pf Test

ITP11002TC1/ TC40 Advantage P.f Malaria Card IR016025 CareStart™Malaria HRP2 (Pf) G0141 diagnosticks- Malaria (Pf)Cassette WB KMFC6001 First Response® Malaria Ag P. falciparum (HRP2) Card Test I13FRC IMMUNOQUICK® MALARIA falciparum 0502_K25 KHB® Malaria Ag P.f Rapid Test KH-R-06-20 05FK50/05FK53 SD BIOLINE Malaria Antigen Pf Vision Malaria Pf VB01 Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25 Advanced Quality™ Rapid Malaria Test (Pf/Pan) ITP11005 Advantage Mal Card IR221025 Advantage Malaria Pan + Pf Card IR231025 BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) C30RHA25 BioTracer™ Malaria Pf/PAN Rapid Card 17012 CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo G0131 Clearview® Malaria Dual VB20 DIAQUICK Malaria P.f/Pan Cassette Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat EzDx™ Malaria Pan/Pf Rapid Test Detection Kit RK MAL 001 Serums & Vaccines Ltd. ) First Response® Malaria Ag. pLDH/HRP2 Combo Card Test I16FRC Premier Medical Corporation GenBody™Malaria Pf/Pan Ag MALAG100 GenBody Inc. Genedia® Malaria P.f/Pan Ag Rapid Test 20-0146-01 Green Cross Medical Science Corp. (Korea) Humasis Malaria Pf/Pan Antigen Test AMAL-7025 Humasis Co., Ltd. ICT Malaria Dual Test ML03 ICT INTERNATIONAL Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit A03-18-322 Artron Laboratories Inc. Malaria Pf/Pan One Step Rapid Test RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. Malaria pf (pLDH) / PAN-pLDH Test Device MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd MD Malaria Pf/Pan(pLDH) test NG-MALSARL NG Biotech, Z.A. NG-Test MALARIA Pf/Pan (pLDH) W23-001 OnSite Pf/Pan Ag Rapid Test R0113C CTK Biotech Inc. Parascreen® Rapid test for malaria Pan/Pf 50310025 Zephyr Biomedicals

(continued)

Results

51

52

Table 6: Heat stability testing results for 42 malaria RDTs on a cultured P. falciparum sample at low (200) and high (2000) parasite density (parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °Ca (continued) Positive test results for P. falciparum (Pf line) Positive test results for P. falciparum (pan line) 200 parasites/μL Baseline Number of tests positive (max. 30) Lots 1 and 2 combined 0.0 0.0 0.0 NA NA NA NA NA NA 11.0 30.0 20.0 30.0 18.0 30.0 NA NA NA NA 10.0 10.0 NA NA NA NA NA NA NA NA NA NA 10.0 10.0 10.0 10.0 10.0 9.0 10.0 10.0 10.0 10.0 NA NA 6.0 0.0 0.0 30.0 0.0 0.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 7.0 10.0 10.0 9.0 10.0 NA NA

Positive test results for P. falciparum (Pf line) 2000 parasites/μL Baseline Number of tests positive (max. 10) Lots 1 and 2 combined 35 °C 45 °C Baseline

Positive test results for P. falciparum (pan line) 2000 parasites/μL 35 °C 45 °C Number of tests positive (max. 10) Lots 1 and 2 combined 10.0 10.0 6.0 NA NA NA NA NA NA 10.0 10.0 6.0 10.0 6.0 NA NA NA NA NA NA 10.0 10.0 10.0 10.0 0.0 NA NA NA NA NA NA 9.0 10.0

Product Baseline Number of tests positive (max. 30) Lots 1 and 2 combined 30.0 30.0 30.0 30.0 25.0 30.0 30.0 30.0 30.0 NA NA NA NA NA NA 29.0 28.0 29.0 29.0 29.0 27.0 30.0 30.0 19.0 25.0 30.0 30.0 30.0 30.0 29.0 30.0 30.0 29.0

Catalogue number

Manufacturer 35 °C 45 °C 35 °C 45 °C

200 parasites/μL

IMPN-C52 Hangzhou Biotest Biotech Co. Ltd. 05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc.

AM4650-K GM006 MAT-PF/PAN-50 523352

55IC402-50

RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a Positive results presented in the table are based on stability of a positive reader 1 result

Figure 20: Heat stability of P. falciparum-specific test line of P. falciparum-only tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation No. of positive results from two lotsa 30 Advantage P.f Malaria Card - IR016025 CareStartTMMalaria HRP2 (Pf) - G0141 diagnosticks-Malaria (Pf) Cassette WB - KMFC6001

20

First Response® Malaria Ag P. falciparum (HRP2) Card Test - I13FRC Immunoquick Malaria falciparum - 0502_K25 KHB® Malaria Ag P.f Rapid Test - KH-R-06-20 SD BIOLINE Malaria Ag Pf - 05FK50/05FK53

10

Vision Malaria Pf - VB01 Advanced QualityTM One Step Malaria Pf Test - ITP11002TC1/TC40

0 a Maximum

Baseline

35 °C

45 °C

score is 30 (15 tests x 2 lots)

Figure 21: Heat stability of P. falciparum-specific test line of P. falciparum-only tests against a high-density P. falciparum sample (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation. No. of positive results from two lotsa 10 8 6 4 2 0 a Maximum

Advantage P.f Malaria Card - IR016025 CareStartTM Malaria HRP2 (P f) - G0141 diagnosticks-Malaria (Pf) Cassette WB - KMFC6001 First Response ® Malaria Ag P. falciparum (HRP2) Card Test - I13FRC Immunoquick Malaria falciparum - 0502_K25 SD BIOLINE Malaria Ag Pf - 05FK50/05FK53 Vision Malaria Pf - VB01 Advanced QualityTM One Step Malaria Pf Test - ITP11002TC1/TC40 KHB ® Malaria Ag P.f Rapid Test - KH-R-06-20

Baseline

35 °C

45 °C

score is 10 (5 tests x 2 lots);

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 53

Figure 22: Heat stability of P. falciparum-specific test line in combination tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation. Advantage Malaria Pan + Pf Card - IR231025 Clearview® Malaria Dual - VB20 Ez DxTM Malaria Pan/Pf Rapid Test Detection Kit - RK MAL 001 First Response® Malaria pLDH/HRP2 Combo Test - I16FRC ICT Malaria Dual Test - ML03 Humasis Malaria P.f/Pan Antigen Test - AMAL-7025 Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test - MAT-PF/PAN-50 MD Malaria Pf/Pan(pLDH) test - MDMALLDH001 NG-Test MALARIA Pf/Pan (pLDH) - NG-MAL-W23-001 One Step Malaria P.F/P.V Test - 523352 OnSite Pf/Pan Ag Rapid Test - R0113C Parascreen® Rapid test for malaria Pan/Pf - 50310025 RightSignTM Malaria P.f./Pan Rapid Test Cassette - IMPN-C52 SD BIOLINE Malaria Ag P.f/Pan - 05FK60/05FK63 BIOCREDI T Malaria Ag Pf/Pan (HRPII/pLDH) - C30RHA25 CareStartTM Malaria HRP2/pLDH (P f/PAN) Combo - G0131 DIAQUICK Malaria P.f/Pan Cassette - Z11200CE Malaria Pf/Pan One Step Rapid Test - RT 20222 GenBodyTM Malaria Pf/Pan Ag - MALAG100 ParaHIT® fV Rapid test for P. falciparum and P.vivax Malaria - Device - 55IC402-50 Vikia ® Malaria Ag Pf/Pan - 412499 Wondfo® One Step Malaria P.f/P.v Whole Blood Test - W056-C BioTracerTM Malaria Pf/PAN Rapid Card - 17012 ASAN E asy Test® Malaria Pf/Pan Ag - AM4650-K Genedia ® Malaria P.f/Pan Ag Rapid Test - 20-0146-01 Advanced QualityTM Rapid Malaria Test (Pf/Pan) - ITP11005 Malaria Pf (HRPII)/ PV (P LDH) Antigen Detection Test Device - GM006 ACCUCARE ONE STE P MALARIA Pf/Pan Antigen Test - MAGC 25 Malaria pf (pLDH) / PAN-pLDH Test Device - MFV -124 Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit - A03-18-322 Advantage Mal Card - IR221025

No. of positive results from two lotsa

30

20

10

0

Baseline

35 °C

45 °C

a Maximum

score is 30 (15 tests x 2 lots)

Figure 23: Heat stability of P. falciparum-specific test line in combination tests against a high-density P. falciparum sample (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation. ACCUCARE ONE STE P MALARIA Pf/Pan Antigen Test - MAGC 25 Advanced QualityTMRapid Malaria Test (Pf/Pan) - ITP11005 Advantage Mal Card - IR221025 Advantage Malaria Pan + Pf Card - IR231025 ASAN E asy Test® Malaria Pf/Pan Ag - AM4650-K BioTracerTMMalaria Pf/PAN Rapid Card - 17012 CareStartTMMalaria HRP2/pLDH (P f/PAN) Combo - G0131 Clearview® Malaria Dual - VB20

No. of positive results from two lotsa

10 8 6 4 2 0

DIAQUICK Malaria P.f/Pan Cassette - Z11200CE Ez DxTMMalaria Pan/Pf Rapid Test Detection Kit - RK MAL 001 First Response® Malaria pLDH/HRP2 Combo Test - I16FRC GenBodyTMMalaria Pf/Pan Ag - MALAG100 Genedia ® Malaria P.f/Pan Ag Rapid Test - 20-0146-01 Malaria pf (pLDH) / PAN-pLDH Test Device - MFV -124 Humasis Malaria P.f/Pan Antigen Test - AMAL-7025 Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit - A03-18-322 Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test - MAT-PF/PAN-50 MD Malaria Pf/Pan(pLDH) test - MDMALLDH001 NG-Test MALARIA Pf/Pan (pLDH) - NG-MAL-W23-001 One Step Malaria P.F/P.V Test - 523352 OnSite Pf/Pan Ag Rapid Test - R0113C Parascreen® Rapid test for malaria Pan/Pf - 50310025 RightSignTMMalaria P.f./Pan Rapid Test Cassette - IMPN-C52 Vikia ® Malaria Ag Pf/Pan - 412499 Wondfo® One Step Malaria P.f/P.v Whole Blood Test - W056-C BIOCREDI T Malaria Ag Pf/Pan (HRPII/pLDH) - C30RHA25 ParaHIT® fV Rapid test for P. falciparum and P.vivax Malaria - Device - 55IC402-50 SD BIOLINE Malaria Ag P.f/Pan - 05FK60/05FK63 Malaria Pf/Pan One Step Rapid Test - RT 20222 ICT Malaria Dual Test - ML03 Malaria Pf (HRPII)/ PV (P LDH) Antigen Detection Test Device - GM006

Baseline

35 °C

45 °C

a Maximum

score is 10 (5 tests x 2 lots)

54

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Figure 24: Heat stability of pan line of pan-specific tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation. No. of positive results from two lotsa 30 CareStartTM Malaria pLDH (P AN) - G0111 Advantage Pan Malaria Card - IR013025

20

10

0 a Maximum

Baseline

35 °C

45 °C

score is 30 (15 tests x 2 lots)

Figure 25: Heat stability of pan line of pan-specific tests against a high-density (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation. No. of positive results from two lotsa 10 8 6 4 2 0 a Maximum

CareStartTMMalaria pLDH (PAN) - G0111 Advantage Pan Malaria Card - IR013025

Baseline

35 °C

45 °C

score is 10 (5 tests x 2 lots)

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 55

Figure 26: Heat stability of pan line of combination tests against a low-density P. falciparum sample (200 parasites/μL). Positivity rate at baseline and after 60 days’ incubation. Advantage Malaria Pan + Pf Card - IR231025 CareStartTM Malaria HRP2/pLDH (P f/PAN) Combo - G0131 Malaria pf (pLDH) / PAN-pLDH Test Device - MFV-124 Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit - A03-18-322 BioTracerTM Malaria Pf/PAN Rapid Card - 17012 Genedia ® Malaria P.f/Pan Ag Rapid Test - 20-0146-01 ACCUCARE ONE STE P MALARIA Pf/Pan Antigen Test - MAGC 25 Clearview® Malaria Dual - VB20 Ez DxTM Malaria Pan/Pf Rapid Test Detection Kit - RK MAL 001 OnSite Pf/Pan Ag Rapid Test - R0113C First Response® Malaria pLDH/HRP2 Combo Test - I16FRC BIOCREDI T Malaria Ag Pf/Pan (HRPII/pLDH) - C30RHA25 RightSignTMMalaria P.f./Pan Rapid Test Cassette - IMPN-C52 Parascreen® Rapid test for malaria Pan/Pf - 50310025 NG-Test MALARIA Pf/Pan (pLDH) - NG-MAL-W23-001 Advanced QualityTM Rapid Malaria Test (Pf/Pan) - ITP11005 Advantage Mal Card - IR221025 DIAQUICK Malaria P.f/Pan Cassette - Z11200CE GenBodyTM Malaria Pf/Pan Ag - MALAG100 ICT Malaria Dual Test - ML03 Humasis Malaria P.f/Pan Antigen Test - AMAL-7025 Malaria Pf/Pan One Step Rapid Test - RT 20222 MD Malaria Pf/Pan(pLDH) test - MDMALLDH001 SD BIOLINE Malaria Ag P.f/Pan - 05FK60/05FK63 Vikia ® Malaria Ag Pf/Pan - 412499

No. of positive results from two lotsa

30

20

10

0

Baseline

35 °C

45 °C

a Maximum

score is 30 (15 tests x 2 lots)

Figure 27: Heat stability of pan line of combination tests against a high-density P. falciparum sample (2000 parasites/μL). Positivity rate at baseline and after 60 days’ incubation. Advantage Mal Card - IR221025 Advantage Malaria Pan + Pf Card - IR231025 BioTracerTM Malaria Pf/PAN Rapid Card - 17012 CareStartTM Malaria HRP2/pLDH (P f/PAN) Combo - G0131 Ez DxTM Malaria Pan/Pf Rapid Test Detection Kit - RK MAL 001 First Response® Malaria pLDH/HRP2 Combo Test - I16FRC Humasis Malaria P.f/Pan Antigen Test - AMAL-7025 MD Malaria Pf/Pan(pLDH) test - MDMALLDH001 NG-Test MALARIA Pf/Pan (pLDH) - NG-MAL-W23-001 Parascreen® Rapid test for malaria Pan/Pf - 50310025 SD BIOLINE Malaria Ag P.f/Pan - 05FK60/05FK63 BIOCREDI T Malaria Ag Pf/Pan (HRPII/pLDH) - C30RHA25 OnSite Pf/Pan Ag Rapid Test - R0113C Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit - A03-18-322 Malaria Pf/Pan One Step Rapid Test - RT 20222 Clearview® Malaria Dual - VB20 Malaria pf (pLDH) / PAN-pLDH Test Device - MFV-124 DIAQUICK Malaria P.f/Pan Cassette - Z11200CE ICT Malaria Dual Test - ML03 Advanced QualityTM Rapid Malaria Test (Pf/Pan) - ITP11005 ACCUCARE ONE STE P MALARIA Pf/Pan Antigen Test - MAGC 25 RightSignTM Malaria P.f./Pan Rapid Test Cassette - IMPN-C52 GenBodyTM Malaria Pf/Pan Ag - MALAG100 Vikia ® Malaria Ag Pf/Pan - 412499 Genedia ® Malaria P.f/Pan Ag Rapid Test - 20-0146-01

No. of positive results from two lotsa

10 8 6 4 2 0

Baseline

35 °C

45 °C

a Maximum

score is 10 (5 tests x 2 lots)

56

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

14. Ease-of-use DESCRIPTION and RDT anomalies After becoming proficient in using a product, two technicians produced a joint agreed assessment of product usability. The results, which are a description of the product with emphasis on aspects considered important for ease of use in the field, are presented in Table 7. It is important to note that the assessment does not include a comparison of blood transfer devices, which are critical to both the safety and the accuracy of the testing procedure and pose a significant challenge to many users. These may vary for manufacturers with many products. Procurement decisions should be based on which transfer devices are best suited for the intended users, and this should be discussed with the manufacture before procurement. It is strongly recommended that RDT packaging, contents, safety and ease of use be assessed in the field as part of the product selection process (Annex S2, Table AS2.1). In round 5, technicians regularly recorded specific observations (or anomalies) based on a list of problems encountered with some production lots evaluated in past rounds of testing and at WHO-FIND lot testing laboratories. Since March 2012, these observations have been included in all WHO-FIND lot testing reports and were recorded as part of product testing for the first time in round 5. Table 8 shows the percentages of products for which anomalies were observed in at least one of the RDTs tested; it does not give the frequency of the observation for a given product. Generally, users should be aware of major anomalies that may be encountered in production lots (Fig. AS2.1), as they can affect interpretation of RDT results.

Table 8: Observations on RDT production lots that might affect interpretation of the results Observations/anomalies Red background Incomplete clearing Failure to flow Shift or misplacement of strip Ghost lines Diffuse test lines Patchy broken test line No.(%) of products with at least one recorded observation 42 (100) 42 (100) 26 (61.9) 10 (23.8) 7 (16.7) 2 (4.8) 2 (4.8)

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Results 57

58

Table 7: Ease-of-use description of 42 malaria RDTs included in round 5: WHO malaria RDT product testing Blood safetya Language of instruction Instruction qualityb

Product

Catalogue number

Manufacturer

Com- Number Total Blood Diagram bined of timed time to transfer Format Mixing Retract- Strip Diagram of result Score No Score wells able exposed (max. 3) diagram of result & method (max. 2) score (max. 5) steps result device involved needle (1) (2) 1 1 1 1 1 1 1 1 1 0 1 2 – – X 1 2 4 0 1 2 – – X 1 2 4 15 20 1 1 3 – – X 1 15 2 5 0 0 1 – – X 1 15 2 3 None Pipette 0 1 2 – – X 1 20 Pipette 2 4 0 1 2 – X – 1 20 Loop 1 3 Cassette Cassette Dipstick Cassette Inverted Cassette cup Pipette Cassette 0 1 2 – – X 1 20 Pipette Cassette 2 4 0 1 2 – – X 1 20 Pipette Cassette 2 4 English English English English English English English, French English 1 1 3 – – X 1 15 English 2 5 Capillary Cassette tube

Items included in packagec

Pf only

Advanced Quality™ One Step Malaria Pf Test

ITP11002TC1/ TC40

InTec Products, Inc.

Advantage P.f Malaria Card

IR016025

J. Mitra & Co. Pvt. Ltd.

CareStart™ Malaria HRP2 (Pf)

G0141

Access Bio. Inc.

diagnosticks- Malaria (Pf)Cassette WB

KMFC6001

First Response® Malaria Ag P. falciparum I13FRC (HRP2) Card Test

SSA Diagnostics & Biotech Systems Premier Medical Corporation

IMMUNOQUICK® MALARIA falciparum

0502_K25

BIOSYNEX

KHB® Malaria Ag P.f Rapid Test

KH-R-06-20

Shanghai Kehua Bioengineering Co.,Ltd.

SD BIOLINE Malaria Antigen Pf

05FK50/05FK53 Standard Diagnostics Inc.

Vision Malaria Pf

VB01

Vision Biotech (Pty) Ltd

Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator) Test tube, buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (colour indicator) Buffer, alcohol swab, lancet, dessicant (colour indicator) 1 1 20 15 Pipette Cassette Capillary Cassette tube English English None None

MAGC 25 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 0 1 2 2 3 2 2 0 1 2 0 1 2 – – – – – – – 0 1 2 – – – – – X – – – 0 1 2 – – 0 1 2 – – 0 1 2 – – X X X X X X X – X X X 0 1 2 – – X 2 2 2 2 2 2 2 2 1 2 2 2 0 1 2 – – X 2 0 1 2 – – X 2 4 4 4 4 4 4 4 4 4 4 3 5 4 4

LAB-CARE Diagnostics (India) PVT. LTD. 1 0 1 2 – – X 2 4

Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan)

ITP11005

InTec Products, Inc.

Advantage Mal Card

IR221025

J. Mitra & Co. Pvt. Ltd.

1 1 1 1 1 1 1 1 1 1 1 1 1

20 20 30 20 20 25 20 20 20 30 15 30 30

Pipette Pipette

Cassette Cassette

English English English English Pipette Pipette Loop Pipette Pipette Loop Loop Loop Cassette Cassette Cassette Cassette Cassette Cassette Cassette Cassette Inverted Cassette cup English English English, German English English English English English, French, Spanish English

Advantage Malaria Pan + Pf Card

IR231025

J. Mitra & Co. Pvt. Ltd.

Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator) None

BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) C30RHA25

RapiGEN INC.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) Capillary Cassette tube Loop Cassette None

17012

Bio Focus Co., Ltd.

BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo

G0131

Access Bio. Inc.

Clearview® Malaria Dual

VB20

Orgenics Ltd.(IS)

DIAQUICK Malaria P.f/Pan Cassette

Z11200CE

None Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (colour indicator) None

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag

I16FRC

MALAG100

Genedia® Malaria P.f/Pan Ag Rapid Test

20-0146-01

DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea)

Humasis Malaria Pf/Pan Antigen Test

AMAL-7025

Humasis Co., Ltd.

ICT Malaria Dual Test

ML03

ICT INTERNATIONAL

Buffer, alcohol swab, lancet, dessicant (no colour indicator) None Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (colour indicator)

Table 7 (continued) Blood safetya Language of instruction English English English English English English English English English None None

Instruction qualityb Items included in packagec

Product

Catalogue number 1 1 1 1 1 1 1 1 1 0 1 2 – – X 1 15 2 4 Inverted Cassette cup Pipette Cassette 0 1 2 – – X 1 10 Pipette Cassette 2 4 0 1 2 – X – 1 20 Loop Cassette 1 3 0 1 2 – – X 1 30 Loop Cassette 2 4 0 1 2 – – X 1 30 Pipette Cassette 2 4 0 1 2 – – X 1 30 Pipette Cassette 2 4 0 1 2 – – X 1 20 None Cassette 2 4 0 1 2 – – X 1 20 Pipette Cassette 2 4 0 1 2 – – X 1 30 None Cassette 2 4

Manufacturer

Com- Number Total Blood Diagram bined of timed time to transfer Format Mixing Retract- Strip Diagram of result Score No Score wells able exposed (max. 3) diagram of result & method (max. 2) score (max. 5) steps result device involved needle (1) (2)

Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit A03-18-322

Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. Malaria pf-LDH/PAN-LDH Antigen Test Device MFV-124 AZOG, INC. Medical Diagnostech MD Malaria Pf/Pan(pLDH) test MDMALLDH001 (Pty) Ltd NGNG-Test MALARIA Pf/Pan (pLDH) SARL NG Biotech, Z.A. MAL-W23-001 R0113C CTK Biotech Inc. OnSite Pf/Pan Ag Rapid Test

Malaria Pf/Pan One Step Rapid Test

RT 20222

Parascreen® Rapid test for malaria Pan/Pf 50310025

Zephyr Biomedicals

None Buffer, alcohol swab, lancet, dessicant (colour indicator) Buffer, alcohol swab, lancet, dessicant (colour indicator) None Buffer, alcohol swab, lancet, dessicant (colour indicator) None Buffer, alcohol swab, lancet, dessicant (colour indicator)

RightSign™ Malaria P.f./Pan Rapid Test Cassette

IMPN-C52

Hangzhou Biotest Biotech Co. Ltd.

SD BIOLINE Malaria Antigen Pf/Pan

05FK60/05FK63 Standard Diagnostics Inc.

Vikia® Malaria Ag Pf/Pan

412499

IMACCESS S.A.S

1

0

1

2 – – X 1 20

2

4

Danish, English, French, German, None Greek, Italian, Polish, Portuguese, Spanish, Swedish Loop Cassette English None

Pf and Pv/Pvom 1 1 1 1 1 1 0 1 2 – – X 2 1 1 3 – – X 2 0 1 2 – – X 2 4 5 4 0 1 2 – – X 2 4 0 1 2 – – X 2 4 1 1 1 1 1 0 1 2 – – X 1 2 4 30 20 20 15 25 15

ASAN Easy Test® Malaria Pf/Pan Ag

AM4650-K

GM006

Pipette Pipette None

Cassette Cassette Cassette Capillary Cassette tube Pipette Cassette

English English English English English

Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test

MAT-PF/ PAN-50

Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (colour indicator) None Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator)

One Step Malaria P.F/P.V Test (Cassette)

523352

ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd.

Span Diagnostics Ltd.

ParaHIT®fV Rapid test for P. falciparum 55IC402-50 and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole W056-C Blood Test Pan only 1 1 0 1 2 – – 0 1 2 – – X X 2 2

Guangzhou Wondfo Biotech Co. Ltd.

Advantage Pan Malaria Card

IR013025

J. Mitra & Co. Pvt. Ltd.

4 4

1 1

20 20

Pipette Pipette

Cassette Cassette

English English

CareStart™ Malaria pLDH (PAN)

G0111

Access Bio. Inc.

Buffer, alcohol swab, lancet, dessicant (no colour indicator) Buffer, alcohol swab, lancet, dessicant (no colour indicator)

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

a

Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae Mixing wells involved: Yes=0; No=1; retractable needle: yes=1; no=0; strip exposed, not within card or cassette: exposed=0, covered=1 b No diagrams=0; diagram of results=1; diagram of result and method=2 c These are items in addition to the test device and blood transfer device and are not necessarily standard contents. Procurers should verify what materials accompany test kits with the manufacturer and ensure they procure all the required accessories at the same time.

Results

59

15. Discussion of key findings This report describes the performance of many of the available malaria antigen-detecting RDTs manufactured under the ISO 13485:2003 quality standard. Malaria RDTs can greatly improve the management of febrile illness in malaria-endemic areas. To be useful in this context, they must have adequate: • sensitivity, to detect nearly all clinically significant cases of malaria; • specificity, to accurately discriminate non-malarial febrile illness from malaria, to ensure appropriate management and accurate disease monitoring; • stability, to maintain accuracy after transport and storage in ambient conditions; and • ease-of-use and safety, to allow safe, correct preparation and accurate interpretation of results. Malaria RDTs were evaluated in terms of these four major requirements in order to assist national malaria control programmes and other procurement agencies in selecting products appropriate for their needs. The panel used allowed successful discrimination between the RDTs evaluated, which had a considerable range of performance. A number of products showed a high rate of antigen detection combined with a low false-positive rate and good heat (thermal) stability. These attributes are essential if the tests are to be relied upon as a basis for decisions about malaria treatment in most endemic populations. Overall, the mean product PDS against low-density P. falciparum samples in round 5 was 81.0%, consistent with the results of round 4 (81.6%) and suggesting that performance may be plateauing after several rounds of improvement.1 For P. vivax, the mean PDS of 61.3% is the highest achieved.2 The median false-positive rate was 1.3%, which is comparable with that in previous rounds; however, in rounds 4 and 5, a limited number of products returned exceptionally high false-positive rates. Overall, a high level of performance has been maintained in P. falciparum -only tests and improved performance in P. vivax-detecting RDTs. Ten products tested in round 1 of the programme were resubmitted for testing in round 5. The performance of most of the products was comparable 5 years after the initial testing; only one that had previously fulfilled the WHO procurement criteria no longer does. Another product showed improved performance and fulfilled the WHO procurement criteria when it previously did not. The evaluation is performed against a standardized panel of cultured P. falciparum and frozen blood samples by experienced technicians in a research laboratory and is not 1 PDS for P. falciparum in rounds 1, 2 and 3 was 67.2%, 69.9%, 75.5%,

therefore a field evaluation of the accuracy of RDTs in a specific epidemiological context in the hands of the intended users. The panel is designed to mimic fresh blood samples from actual cases as closely as possible, while allowing direct comparison of a large number of products simultaneously with control for confounding factors and is calibrated to a level likely to discriminate differences in the performance of various products. The discussion points described below should therefore be taken into account in interpreting the results.

15.1.  Panel detection score and its relation to sensitivity Evaluation of the RDTs against the phase-2 wild-type parasite panel with a parasite density of 200 parasites/µL (Figs 10 and 11) revealed a wide range of frequency and consistency of antigen detection between products, recorded as the PDS. As expected, testing at higher parasite density (2000 or 5000 parasites/µL) resulted in smaller differences in performance. As two tests from two different lots were tested at 200 parasites/µL and as all four results had to be positive in order for a sample to be considered detected by an RDT, a positive result indicated the ability of a product to detect the target antigen in the sample and to do this consistently (both tests from both lots). A parasite density of about 200 parasites/µL should be detected to ensure high field sensitivity for clinically significant malaria infection in many malaria-endemic populations (9). The PDS in the panels used in this evaluation differs from the test sensitivity in clinical settings for five main reasons. (i) The performance of different lots or batches of the same product may vary. Variation in lot performance is an issue for all diagnostics; therefore, the results found in the evaluation may not predict the results for subsequent RDT lots. It is important to test lots before their distribution in the field to ensure that the expected performance is maintained (section 15.2). (ii) In clinical settings, patients show wide variation in parasite density, the range depending on the local epidemiology of the disease. The parasite density in the population tested affects the clinical sensitivity of a test. The PDS against a test panel of blood samples diluted to 200 parasites/µL is likely to underestimate the clinical sensitivity of an RDT in areas where symptomatic patients have much higher parasite densities. Many tests that show only moderate detection of the 200-parasites/ µL panel may perform well in such settings, as indicated by the better PDS of most products against the panel at 2000 parasites/µL. The small differences in PDS seen in Figs S1, S2 and 9–11 and Tables 4 and 5 found among the better-performing RDTs in this evaluation are unlikely to result in noticeable differences in clinical sensitivity,

respectively. 2 PDS

for P. vivax in rounds 1, 2, 3 and 4 was 36.0%, 58.9%, 47.1% and 51.3%, respectively.

60

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

and other issues, such as stability, cost, experience and training of the intended users, ease of use (Annex S2) and manufacturing capacity may be equally important factors in test selection. Consideration of the parasite density in target populations and the probable sensitivity of RDTs in the field indicates that, even in areas with high transmission and strong malaria immunity, the population may include individuals with a low parasite density but clinically significant infection (e.g. young children, pregnant women, people who regularly use bed nets, immigrants and people with reduced immunity). The ability to detect low parasite-density infections reliably, therefore, remains important. As some countries move towards elimination of malaria, population immunity will decrease and/or clinical cases may be detected earlier, and it will become increasingly important to use diagnostic tests that detect low parasite density (i.e. with a high PDS against samples with 200 parasites/µL). (iii) The performance of tests against the challenge panel may not always predict sensitivity in clinical testing, e.g. when antigen expression by certain parasite populations differs greatly from that in the panel. For example, P. falciparum strains in some areas of India and South America do not express HRP2 antigens because of gene deletions (10, 11). If a significant proportion of parasites in a given area do not express HRP2 and HRP3, tests to detect other target antigens (e.g. pLDH or aldolase) must be used. To date, parasite populations with a high frequency of non-expression of target antigens have not been identified elsewhere than South America.1 (iv) The conditions under which RDTs are transported and stored can alter their sensitivity in the field. The tests evaluated in round 5 were shipped and stored under conditions intended to safeguard them from degradation by high temperature or other extreme conditions. If such precautions are not taken with purchased RDTs, loss of performance could result. The ambient temperature of storage conditions varies widely in the settings in which these tests are commonly used, as does the temperature during transport; therefore, the requirements for the heat stability of a product will differ. Tests should be transported and stored well within the temperature range recommended by the manufacturer and extremes of temperature avoided (31, 32). (v) Diagnostic sensitivity and specificity depend on the quality of preparation and interpretation of the tests. Highly trained technicians tested all the products in this evaluation. In clinical settings, malaria RDTs are often used by health workers with limited training and supervision; therefore, simple design and clearly interpretable results are required to ensure translation of the technical proficiency of a product into accurate diagnoses in the field (33).

15.2.  False-positive rate and specificity False-positive rates are reported against a panel of 59 clean-negative samples taken from blood donated in lowtransmission settings by people without symptoms of malaria. In addition, false-positive rates were calculated with a smaller number of samples with specific characteristics that affect the likelihood of a false-positive result from an immunodiagnostic test (e.g. rheumatoid factor, anti-nuclear antibody) or that may be significant in a specific population in a malaria-endemic area (e.g. leishmaniasis, dengue). The importance of these results depends on the intended area of use. High false-positive rates with samples of blood from dengue patients, for example, might not be a significant factor in regions in which dengue does not occur. In view of the small number of samples in each category in this evaluation, the results should be considered primarily a guide to potential cross-reactions, which should be closely monitored if they are relevant to the target population. In general, it is preferable to procure a product with a low rate of false-positive reactions. In the case of many diagnostic tests, a trade-off must be made between a preference for a high rate of antigen detection (sensitivity) and a low false-positive rate (specificity). The context in which the test will be used will guide the relative importance of these two factors in the choice of one product over another. Overall, in this evaluation, there was no correlation between a lower PDS (loss of sensitivity) and a low false-positive rate (high specificity). A number of products had both a high PDS and a low false-positive rate.

15.3. Reactivity of combination HRP2 and pan-pLDH test lines against P. falciparum samples Instructions for the use of P. falciparum/pan and pan/P. falciparum combination tests classify P. falciparum infections as either HRP2 test line-positive alone or in combination with the pan-pLDH line. Combination tests that return only a positive HRP2 test line may be incorrectly interpreted as false-positives for malaria infection secondary to persistent (HRP2) antigenaemia. The results in this report clearly indicate that most combination tests in which HPR2 is used for the detection of P. falciparum return positive results only on the HRP2 band at lower densities of P. falciparum (Table A4.2). When both the HRP2 and the pan test bands were positive, the mean band intensity was significantly lower on the pan test band than on the HRP2 test band. Therefore, it is important to reinforce adherence to the manufacturer’s instructions for use (Annex 2) and to emphasize that for combination HRP2/pan-pLDH tests, a HRP2 test line-positive alone, may well be attributed to the poor reactivity of pan-pLDH lines.

1

Cheng Q, Gatton M, Barnwell J, et al. Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting. Acceptedfor publication.

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Discussion 61

15.4.  Heat (thermal) stability The RDTs evaluated were held for 2 months at room temperature (< 25 °C) and at 35 °C and 45 °C at 75% humidity and tested to evaluate stability at these temperatures. The importance of thermal stability depends on the conditions under which a product will be transported and stored. Thus, stability at high temperatures is vital if an RDT is to be stored at clinics in a country where the ambient temperature can reach 45 °C in the hot season but is less critical in a high-altitude or cooler environment where the temperature rarely rises above 35 °C. Many commercially available RDTs indicate 30 °C or 40 °C as the maximal storage temperature. Higher temperatures were tested in this evaluation because malaria-endemic countries often have maximum ambient temperatures of 35 °C, although use of cool storage can allow storage of products below this temperature. When RDTs are likely to be transported and stored at high ambient temperatures, heat (thermal) stability must be considered a significant factor in ensuring sensitivity. High humidity accelerates the degradation of malaria RDTs and other lateral flow tests. All the products in this evaluation were packaged in individual envelopes containing a desiccant and are designed to be moisture-proof. This allows the user to open the envelope of a test at the time of use, limiting exposure to high humidity. During the stability-testing phase of this evaluation, the RDTs were stored at 75% humidity. The packaging should, if in good condition, protect the contents from exposure to high humidity during storage. The results presented here provide an assessment of both the stability of the RDT and the quality of its packaging. A limitation of our evaluation is that we can assess only the stability of test lines to detect cultured P. falciparum samples. Several products showed high stability at the temperatures and times used in the evaluation. In general, in this round, as in previous ones, pan-specific lines (pLDH) performed less well at baseline and were less stable than HRP2 test lines, so that it was difficult to assess post-incubation stability. While the temperature and humidity were held constant in this evaluation, temperatures in the field fluctuate with the time of day and season. Two months’ storage at a set temperature cannot accurately predict long-term stability under field conditions, but loss of sensitivity for parasite detection over this period indicates that significant sensitivity will be lost if RDTs are stored at similar or higher temperatures for a significant amount of their storage time and the likelihood of greater susceptibility to degradation during short exposure to much higher temperatures, such as during transport (34, 35 ).

15.5. Ease-of-use description and RDT anomalies in production lots The sensitivity and specificity of RDTs depend on the quality of preparation and interpretation. In general, a simpler format with fewer steps or fewer required extraneous materials is likely to be prepared and interpreted more reliably. Thus, cassette-format RDTs are generally more reliably prepared and interpreted than products in dipstick format (36). The extra cost of this format may be offset by the advantages of greater accuracy and, in some cases, less additional equipment required to perform them. The method by which blood is transferred from the patient to the test is important for the safety of the user and for the accuracy of the volume transferred. Devices for blood transfer are supplied with RDTs and vary widely in design and accuracy (30). The performance of blood transfer devices was not formally assessed in this evaluation, as blood was transferred from a tube with a micro-pipette to ensure that the volume specified by the manufacturer was used. Procurement programmes for RDTs should consider the adequacy of the blood transfer device supplied, including the experience of health workers and the cost and time required for retraining. It may be appropriate to discuss with manufacturers the possibility of changing the blood transfer device from that usually supplied. The clarity of results is important for interpreting tests. A clearly visible (intense) test line is less likely to be overlooked than a line that is barely visible. While reading proficiency and adequate workplaces should be ensured, some health workers might have suboptimal vision or work in inadequate lighting. The intensity of the line of the test band was found to be closely associated with the PDS achieved by RDTs (tables A4.2 and A4.3). The importance of format and the simplicity of the test design depend on the intended users. Trained laboratory technicians can handle a complicated procedure more reliably than village volunteers with limited supervision. In all cases, proficiency-based training and adequate supervision should be included in any RDT-based diagnostic programme, and clear instructions should be provided in a language and format appropriate for the user (37, 38). Annex S2 provides guidance on conducting a field-based ease-of-use assessment (Table AS3.1). In the production lots submitted for evaluation, anomalies that affected interpretation of the results were encountered with variable frequency. On the basis of the experience of several rounds of product testing, with 2700 lots tested in the WHO-FIND lot testing programme, a glossary of RDT anomalies has been prepared (Figure AS2.1). This glossary may be used in RDT training programmes to illustrate potential problems with some production lots and how to report them accurately. As many of the anomalies are infrequent, they might not be picked up in manufacturers’ quality control or lot release procedures; therefore, this information is also useful for manufacturers that wish to improve their processes.

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15.6.  Inter-lot variation The testing programme evaluated only two production lots of each product. Malaria RDTs are complex biological products made up of components that are commonly supplied from different sources and are subject to a variety of conditions during manufacture that may affect the quality of the final product. All manufacturers that entered this evaluation provided at least one current ISO 13485:2003 certificate for a manufacturing facility. This standard is designed to ensure consistency in the quality of the final product, if correctly implemented. The results presented here indicate that interlot variation does occur, and WHO strongly recommends that a sample of RDTs from each production lot be tested before their dissemination to the field, to ensure that they meet an appropriate standard. This can be facilitated by WHO through two WHO-recognized lot testing facilities (section 15.2). Inter-test variation, which is also seen, will be detected to some extent by routine lot testing. Ensuring that manufacturers follow good manufacturing standards should minimize the likelihood of inconsistencies due to poor practice in the manufacturing process. Culture-based panels1 that are subsets of the phase-1 panel used in this evaluation are available as reference standards for manufacturers against which to set their lot-release criteria.

the products with the highest PDS for P. falciparum targeted HRP2, a number of pLDH-detecting products had high PDS against P. vivax. The thermal stability of tests that target these different antigens also overlapped for samples with high parasite density. The choice of RDT should take into account the target antigen: HRP2-detecting RDTs should not be used in areas where non-expression of HRP2 is common (10, 11). Tests that detect only HRP2 (without pLDH or aldolase lines) will be of limited use where non-falciparum malaria is common. pLDH (and possibly aldolase) RDTs may have further advantages when antigen persistence (common with HRP2) may result in a high false-positive rate in areas where early retesting in the weeks immediately after treatment is common. As mentioned in section 14.3, however, combination tests with both HRP2 and pan test lines should not be used for discriminating between acute infection and persistent antigenaemia, as the overall reactivity of pan test lines is much lower than that of HRP2 test lines, particularly at low parasite density. The required sensitivity of a test may also vary by species: a less sensitive test may be more acceptable for detection of P. vivax than for P. falciparum, as severe outcomes due to missed diagnoses are less likely. Use of a sufficiently sensitive pan-specific-only test may be appropriate in areas where both P. falciparum and P. vivax occur, if all infections are to be managed initially as P. falciparum infections with artemisinin-based combination therapy, but species-specific monitoring data would be lost. Tests with high PDS for both P. falciparum and P. vivax were found in this and previous rounds of product testing (3–6). Pan-species tests were not evaluated for detection of P. ovale or P. malariae in this evaluation because of lack of sources of suitable mono-species infections with these parasites. Published data suggest that the sensitivity of RDTs for detecting these species is significantly poorer than for P. falciparum and P. vivax (39).

15.7. Target antigens and species The malaria RDTs assessed in this evaluation detect one or more of three parasite antigens, HRP2, pLDH and aldolase, in various combinations. HRP2 is present only in P. falciparum, whereas aldolase and pLDH are present in all four species and may be used as pan or all-species targets. Some tests use differences in pLDH sequences between species as a means to differentiate P. falciparum from P. vivax and other species. There is considerable overlap in the PDS of products that target the different antigens in this evaluation. While

1

To access these panels, contact Malaria_rdt@who.int, cunninghamj@ who.int or info@finddiagnostics.org.

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Discussion 63

16.  USING RESULTS TO ENSURE HIGHQUALITY DIAGNOSIS IN THE FIELD This report provides data to guide malaria control programmes in selecting products that are likely to perform to a high standard in the context in which the programme operates. Final product selection requires that these data be considered systematically, taking into context the distribution of parasite density in the target population in whom the tests will be used and the experience and training of the intended users. Box 3 lists WHO’s minimum RDT selection criteria, as endorsed by the Malaria Policy Advisory Committee, and tables S2, and 5 are colour-coded to reflect these minimum performance criteria for product selection. A web-based tool for filtering product testing results by various parameters is available and maintained by FIND.1 Furthermore, an algorithm to guide selection is given in Annex S3, and detailed guidance was published by WHO in Good practices for selecting and procuring rapid diagnostic tests for malaria (13) and Universal access to malaria diagnostic testing (14). While malaria RDTs can be used in a number of settings, the greatest impact on public health will be brought about by extending access to accurate, parasite-based diagnoses of malaria to regions and populations where good-quality microscopy-based analysis is impractical to maintain. This will allow implementation of recent WHO recommendations on universal parasite-based diagnosis before antimalarial therapy (2) and currently applies to most people at risk for malaria in endemic countries (1). In many settings where RDTs have been introduced, the true rate of parasitaemia has been found to be considerably lower than expected, so that health systems can reduce wastage of antimalarial medicines and focus on appropriate management of non-malarial causes of fever, including early pneumonia and sepsis. A successful RDT programme must therefore address not only malaria but also the management of other common and severe febrile illnesses that occur locally in the differential diagnosis of malaria if an RDT programme is to have its full potential public health impact. supplies of all the necessary materials (including gloves, sharps disposal containers and supplies required for further case management), training of users, community sensitization and monitoring of diagnostic quality and results. This extends malaria management to management of other febrile diseases and health service delivery systems and requires integration with other health programmes. This report provides information to guide procurement of RDTs within this framework. Factors beyond the performance characteristics reported here, however, must influence procurement decisions. An example of an algorithm, including an ease-of-use assessment, is provided to guide decisions in annexes S2 and S3. Details of implementation will vary widely between programmes, depending on local capacity and needs. Further recommendations on budgeting, planning and implementation can be found in Annex 5 and in the relevant WHO guidance document (14).

16.2. Lot testing As a complement to product testing, WHO and FIND currently support laboratories that perform continued quality assurance of RDTs in the form of lot testing. This programme responds to requests from national malaria programmes, manufacturers and procurement bodies to assess the quality of RDT lots before purchase or, when they arrive in a country, before dispersal to the field and for clinical use. Testing is performed against parasite-positive and -negative panels prepared and characterized in the same way as the panels used in this evaluation. A number of national institutions have also developed this capacity. Lot testing reassures countries that the product they have purchased performs to a high standard before distribution and helps to ensure that manufacturers produce consistently good lots and improve their products. Countries and manufacturers ship 100–150 RDTs to regional, WHO-recognized lot testing centres, where they are evaluated against a small panel of parasites at high and low density and against negative samples (Figure 2). They are subsequently incubated at a temperature close to the manufacturer’s specified storage temperature and retested after 18 months. Initial results are available after 5 days, and definitive results after subsequent retesting. Details of the protocol can be found in the methods manual for lot testing (25). National malaria programmes and procurement agencies are encouraged to participate in the lot-testing programme. To access lot-testing through the WHO-FIND programme, contact Malaria_rdt@who.int or info@finddiagnostics.org at least 2 weeks before RDTs are ready for shipment.

16.1. Beyond procurement Diagnostic tests usually represent the start of a health system intervention, and their use is based on the assumption that appropriate patient management, based on testing, will follow. Thus, successful introduction of RDTs requires careful planning beyond rational procurement to ensure consistent 1

http://www.finddiagnostics.org/programs/malaria-afs/malaria/ rdt_quality_control/product_testing/interactive-guide/index.jsp. An interactive guide designed to short-list tests according to programme needs, based on the performance of tests in rounds 2–5 of the WHO product testing programme, can be found at: http://www.finddiagnostics.org/programs/malaria-afs/malaria/rdt_quality_control/ product_testing/interactive-guide/index.jsp (accessed 27 May 2014).

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17. Conclusions This report adds to the large data set on malaria RDT performance published annually since 2009 (3–6). The producttesting programme continues to be an authoritative source in the field of malaria RDT evaluations in terms of the number of products evaluated and its comprehensiveness. New laboratory methods have been developed and validated to support parasite characterization, and this work has generated new findings on variation in antigen content at similar parasite densities and in the structure and expression of HRP proteins. Publication of the results of past WHO product testing rounds has affected the procurement practices of countries and procurement agencies and contributed to a shift in the malaria RDT market towards better-performing products (1). The report of round 5 adds to the number of well-performing RDTs for which comprehensive performance data are now available and provides updated data on 23 products resubmissions.

18. References 1. World Malaria Report 2013. Geneva: World Health Organization; 2013. 2. Guidelines for the treatment of malaria. 2nd Ed. Geneva: World Health Organization; 2010. 3. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 1 (2008). Geneva: World Health Organization; 2009. 4. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 2 (2009). Geneva: World Health Organization; 2010. 5. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 3 (2010–11). Geneva: World Health Organization; 2011. 6. Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 4 (2012). Geneva: World Health Organization; 2012. 7. Informal consultation on laboratory methods for quality assurance of malaria rapid diagnostic tests. Manila: WHO Regional Office for the Western Pacific; 2004 (RS/2004/GE/26(PHL). 8. Prequalification of in vitro diagnostics. Geneva: World Health Organization (http://www.who.int/diagnostics_ laboratory/evaluations/en/, accessed 16 May 2014). 9. Parasitological confirmation of malaria diagnosis. Report of a WHO technical consultation. Geneva, 6–8 October 2009. Geneva: World Health Organization; 2010. 10. Gamboa D, Ho MF, Bendezu J, et al. A large proportion of P. falciparum isolates in the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests. PLoS One, 2010: 5(1): e8091. 11. Kumar N, Pande V, Bhatt RM, Shah NK, Mishra N, Srivastava B, Valecha N, Anvikar AR: Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test. Acta Trop 2013, 125(1):119-121. 12. Malaria RDT interactive guide. http://www.finddiagnostics.org/programs/malaria-afs/malaria/rdt_quality_ control/product_testing/interactive-guide/index.jsp. 13. Good practices for selecting and procuring rapid diagnostic tests for malaria. Geneva: World Health Organization; 2011. 14. Universal access to malaria diagnostic testing: an operational manual. Geneva: World Health Organization; 2011. 15. Kolaczinski J, Mohammed N, Ali, I, et al. Comparison of the OptiMAL rapid antigen test with field microscopy for the detection of Plasmodium vivax and P. falciparum: considerations for the application of the rapid test in Afghanistan. Ann Trop Med Parasitol 2004; 98(1): 15–20. 16. Richter J, Gobels K, Muller-Stover I, et al. Co-reactivity of plasmodial histidine-rich protein 2 and aldolase on a combined immuno-chromographic-malaria dipstick (ICT) as a potential semi-quantitative marker of high Plasmodium falciparum parasitaemia. Parasitol Res 2004; 94(5): 384–385. 17. Mai Huong NM, Davis TM, Hewitt S, et al. Comparison of three antigen detection methods for diagnosis and therapeutic monitoring of malaria: a field study from southern Vietnam. Trop Med Int Health 2002; 7(4): 304–308. 18. Mason DP, Kawamoto F, Lin K, et al. A comparison of two rapid field immunochromatographic tests to expert microscopy in the diagnosis of malaria. Acta Trop 2002; 82(1): 51–59.

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19. van den Broek I, Hill O, Gordillo F, et al. Evaluation of three rapid tests for diagnosis of P. falciparum and P. vivax malaria in Colombia. Am J Trop Med Hyg 2006; 75(6): 1209–1215. 20. McMorrow M, Masanja MI, Kahigwa E, et al. Challenges in routine implementation and quality control of rapid diagnostic tests for malaria—Rufiji District, Tanzania. Am J Trop Med Hyg 2008; 79(3): 385–390. 21. Wanji S, Kimbi HK, Eyong JE, et al. Performance and usefulness of the Hexagon rapid diagnostic test in children with asymptomatic malaria living in the Mount Cameroon region. Malar J 2008; 7: 89. 22. Willcox ML, Sanogo F, Graz B, et al., Rapid diagnostic tests for the home-based management of malaria, in a high-transmission area. Ann Trop Med Parasitol 2009; 103(1): 3–16. 23. Belizario VY, Pasay CJ, Bersabe MJ, et al. Field evaluation of malaria rapid diagnostic tests for the diagnosis of P. falciparum and non-P. falciparum infections. Southeast Asian J Trop Med Public Health 2005; 36(3): 552–561. 24. WHO-TDR-FIND-CDC. Methods manual for product testing of malaria rapid diagnostic tests (version 5). Geneva: World Health Organization; 2012. 25. WHO-TDR-FIND. Methods manual for laboratory quality control testing of malaria rapid diagnostic tests, version 6 . Geneva: World Health Organization; 2010. 26. Product testing round 5. Information for manufacturers and procurers on progress towards product testing of malaria rapid diagnostic tests. Manila: World Health Organization Regional Office for the Western Pacific; 2012 (http://www.wpro.who.int/malaria/sites/ rdt/who_rdt_evaluation/call_for_testing_round5.html, 11 July, 2014 ) 27. Letter from the World Health Organization to manufacturers. Manila: World Health Organization Regional Office for the Western Pacific; 2012 (http://www. wpro.who.int/sites/rdt/who_rdt_evaluation/call_for_ testing_round5.htm; http://www.finddiagnostics.org/, accessed 23 May 2014). 28. Baker J, Ho MF, Pelecanos A, et al. Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests. Malar J 2010; 9: 129. 29. Desakorn V, Dondrop AM, Silamut K, et al. Stagedependent production and release of histidine-rich protein 2 by Plasmodium falciparum. Trans R Soc Trop Med Hyg 2005; 99(7): 517–524.

30. Hopkins H, Oyibo W, Luchavez J, et al. Blood transfer devices for malaria rapid diagnostic tests: evaluation of accuracy, safety and ease of use. Malar J 2011; 10: 30. 31. Methods for field trials of malaria rapid diagnostic tests. Manila: World Health Organization Regional Office for the Western Pacific; 2009. 32. Transporting, storing and handling malaria rapid diagnostic tests at central and peripheral storage facilities. World Health Organization Regional Office for the Western Pacific, USAID Deliver Project, Foundation for Innovative New Diagnostics, Roll Back Malaria Partnership, President’s Malaria Initiative and UNICEF. Manila: World Health Organization Regional Office for the Western Pacific; 2009. 33. Training materials for malaria RDTs. Geneva: Foundation for Innovative New Diagnostics; 2011 (http://www.finddiagnostics.org/programs/malaria/ find_activities/rdt-job-aids/, accessed 27 May 2014). 34. Jorgensen P, Chanthap L, Rebueno A, et al., Malaria rapid diagnostic tests in tropical climates: the need for a cool chain. Am J Trop Med Hyg 2006; 74(5): 750–754. 35. Chiodini PL, Bowers K, Jorgensen P, et al. The heat stability of Plasmodium lactate dehydrogenase-based and histidine-rich protein 2-based malaria rapid diagnostic tests. Trans R Soc Trop Med Hyg 2007; 101(4): 331–337. 36. Rennie W, Phetsouvanh R, Lupisan S, et al., Minimising human error in malaria rapid diagnosis: clarity of written instructions and health worker performance. Trans R Soc Trop Med Hyg 2007; 101(1): 9–18. 37. Harvey SA, Jennings L, Chinyama M, et al., Improving community health worker use of malaria rapid diagnostic tests in Zambia: package instructions, job aid and job aid-plus-training. Malar J 2008; 7(1): 160. 38. Tavrow P, Knebel E, Cogswell L. Using quality design to improve malaria rapid diagnostic tests in Malawi. In: Operations research results 2000; 1(4). 2000. Bethesda, Maryland: United States Agency for International Development. 39. Heutmekers M, Gillet P, Maltha J, et al. Evaluation of the rapid diagnostic test CareStart pLDH Malaria (Pf-pLDH/panpLDH) for the diagnosis of malaria in a reference setting. Malar J 2012; 11: 204.

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Annexes

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annexes 67

Annex S1: Characteristics of evaluation panels used in rounds 1–5 of WHO malaria RDT product testing, 2008–2013 Currently, the basis for diagnosing malaria with antigendetecting RDTs is the detection in a patient’s blood of one or more target malaria antigens, including HRP2 (P. falciparum only), pLDH (Plasmodium spp.(pan-pLDH), P. falciparum (Pf-pLDH), non-falciparum (Pv-pLDH, Pvom-pLDH) and aldolase (all Plasmodium spp). The antigen concentration in samples with the same parasite density varies. Therefore, the concentrations of malaria antigens in the samples that comprise evaluation panels must be consistent in successive rounds of WHO malaria RDT product testing to ensure that the results of each round are highly comparable (statistically equivalent). Therefore, antigen concentrations were quantified in triplicate in all panel samples, including dilution pairs of 200 and 2000 parasites/µL, by quantitative ELISA. Only results that were consistent in the triplicate runs and showed a value factor between the 200 and the 2000 parasites/µL dilutions close to 10 were considered acceptable and eligible for the performance evaluation panel. In some instances, the antigen concentration was below the detection limit of the ELISA, particularly for aldolase, which is present in malaria parasite samples at much lower concentrations than the other two antigens. Samples that gave inconsistent results for more than one of the three antigens were excluded from the panel. Despite careful standardization of procedures, the tables and figures below show a wide variation in antigen concentrations for the same parasite density. There are a number of possible explanations, including differences in the level of antigen expression by isolates; different durations of infection (accumulating antigens); different parasite growth stages at the time of collection (expressing different levels of antigen); the presence of circulating HRP2 from previous growth cycles; and HRP2 produced by parasites sequestered in the host’s vascular tissues that cannot be accounted for in the estimate of parasite density on the blood slide. Before each round of WHO malaria RDT product testing, the distribution of HRP2, pLDH and aldolase concentrations at 200 parasites/µL dilution of the wild-type P. falciparum and wild-type P. vivax samples selected for the phase-2 panels were systematically compared with those in the previous round to ensure there was no statistically significant difference. The figures and tables below show the distribution of antigen concentrations in all five performance evaluation panels. No statistically significant differences were seen (Kruskal-Wallis test; p > 0.15), confirming that the results of each new round are additive (and comparable) to the previous ones. In the following box and whisker plots, the end of whiskers represent minimum and maximum values; the box represents middle 50% of data and the line through box represents median values; the crosses represent the mean values.

Figure AS1.1: Box-and-whisker plot of distribution of P. falciparum HRP2 concentration (ng/mL) in product testing phase 2 (wildtype) panels. Round 5 Round 4 Round 3 Round 2 Round 1 0.5 1 2 4 8 16 32 64 128

P. falciparum HRP2 concentration (ng/ml)

Figure AS1.2: Box-and-whisker plot of distribution of P. falciparum pLDH concentration (ng/mL) in product testing phase 2 (wildtype) panels. Round 5 Round 4 Round 3 Round 2 Round 1 25 12 0. 0. 1 2 4 8 16 32 64

0.

5

P. falciparum pLDH concentration (ng/ml)

Figure AS1.3: Box-and-whisker plot of distribution of P. vivax pLDH concentration (ng/mL) in product testing phase 2 (wild-type) panels. Round 5 Round 4 Round 3 Round 2 Round 1 1 2 4 8 16 32 64

P. vivax pLDH concentration (ng/ml)

Figure AS1.4: Box-and-whisker plot of distribution of P. falciparum aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels. Round 5 Round 4 Round 3 Round 2 Round 1 56 0. 06 0. 25 1 4 16

25 01 0.

P. falciparum aldolase concentration (ng/ml)

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

25

5

Figure AS1.5: Box-and-whisker plot of distribution of P. vivax aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels. Round 5 Round 4 Round 3 Round 2 Round 1 1 2 4 8 16 P. vivax aldolase concentration (ng/ml)

Table AS1.1: Statistics for P. falciparum HRP2 concentration (ng/mL) in product testing phase 2 (wild-type) panels. Round 1 Number of Minimum 25% percentile Median 75% percentile Maximum Mean Std. Deviation a

Round 2 99 0.62 1.90 6.76 16.91 73.70 12.70 15.75

Round 3 99 0.62 2.10 6.83 17.37 66.70 12.77 15.19

Round 4 98 0.62 2.97 6.98 15.65 62.48 12.72 14.72

Round 5 99 0.62 3.00 7.05 15.31 62.48 11.74 13.20

valuesa

78 0.80 2.90 9.57 18.94 73.70 15.28 16.98

The number of values is the number of samples for which consistent ELISA results were obtained.

Table AS1.2: Statistics for P. falciparum pLDH concentration (ng/mL) in product testing phase 2 (wild-type) panels. Round 1 Number of Minimum 25% percentile Median 75% percentile Maximum Mean Std. Deviation a

Round 2 93 0.19 6.27 10.31 20.10 47.15 13.71 10.90

Round 3 92 0.19 6.23 11.18 22.70 47.15 15.08 11.72

Round 4 92 0.19 6.20 10.92 21.28 53.53 14.97 11.98

Round 5 94 0.19 6.90 12.24 23.05 43.02 15.53 11.43

valuesa

74 0.71 6.68 11.95 23.75 47.15 15.31 11.47

The number of values is the number of samples for which consistent ELISA results were obtained.

Table AS1.3: Statistics for P. vivax pLDH concentration (ng/mL) in wildtype product testing phase 2 (wild-type) panels. Round 1 Number of valuesa Minimum 25% percentile Median 75% percentile Maximum Mean Std. Deviation a

Round 2 37 1.64 8.40 17.00 29.69 47.90 20.24 13.27

Round 3 33 1.64 7.30 19.78 31.89 47.90 20.99 13.55

Round 4 32 1.64 6.96 17.50 29.84 47.90 20.00 13.00

Round 5 34 1.64 6.26 13.22 23.42 47.90 16.84 12.59

20 5.10 8.10 12.65 27.40 44.40 17.38 11.57

The number of values is the number of samples for which consistent ELISA results were obtained.

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Annexes 69

Table AS1.4: Statistics for P. falciparum aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels. Round 1 Number of Minimum 25% percentile Median 75% percentile Maximum Mean Std. Deviation a

Round 2 98 0.00 0.74 1.49 2.25 9.90 1.79 1.66

Round 3 99 0.00 0.67 1.40 2.23 9.90 1.76 1.69

Round 4 98 0.00 0.63 1.25 2.25 9.08 1.72 1.68

Round 5 99 0.00 0.52 1.17 2.07 7.74 1.52 1.52

valuesa

77 0.00 0.84 1.61 2.25 9.90 1.93 1.73

The number of values is the number of samples for which consistent ELISA results were obtained.

Table AS1.5: Statistics for P. vivax aldolase concentration (ng/mL) in product testing phase 2 (wild-type) panels. Round 1 Number of Minimum 25% Percentile Median 75% Percentile Maximum Mean Std. Deviation a

Round 2 40 1.70 4.11 6.15 8.47 13.40 6.81 3.15

Round 3 34 1.70 4.07 6.10 8.32 13.30 6.45 2.90

Round 4 33 1.70 4.41 6.16 9.10 15.00 6.86 3.23

Round 5 35 3.21 5.55 6.86 9.43 15.00 7.78 3.30

valuesa

20 3.21 4.02 6.33 8.47 13.15 6.73 2.89

The number of values is the number of samples for which consistent ELISA results were obtained.

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annex S2: Malaria RDT field assessment and anomalies The purpose of this assessment, on a limited number of RDTs, is to assess aspects of packaging, safety and ease-of-use and not to evaluate diagnostic accuracy. Obtain samples of each malaria RDT under consideration (at least one box packaged as intended for delivery to end users). Obtain malaria parasite-negative blood samples, and where readily accessible, parasite-positive blood samples for testing against RDTs. Table AS2.1: Field assessment of RDT packaging, safety and ease-of-use to guide product selection Date of assessment Commercial name Catalogue number Lot number(s) Yes Packaging and accessories The RDT box is in good condition RDTs are in individual sealed pouches The correctly indicated number of RDTs are in the box A desiccant is included in each individual RDT pouch An expiry date is visible on each RDT pouch All required accessories are included in the correct quantities (RDT, buffer, blood transfer device, alcohol swab, lancet, gloves, test tubes (for dipsticks, only) Instructions Instructions are included Instructions are in the national language(s) The instructions are for the correct product The instructions include figures displaying all possible interpretations of the RDT results The text and figures are accurate and consistent (specifically order of test lines and results interpretation) Preparation and procedure The test pouch is easy to open It is easy to write on the test device The test lines on the device are clearly labelled It is easy to use the device for blood collection It is easy to open the buffer bottle or ampoule The buffer bottle or ampoules have sufficient volume for testing all RDTs in the box The buffer bottle or ampoule dispenses even drops It is easy to fill the sample well correctly with the provided blood transfer device It is easy to fill the buffer well correctly (no overflow) The buffer and sample flow well along the test strip Result interpretation Control and test lines Control line is clear Test line(s) are clear Good clearance of blood by time of reading Steps and reading time Reading time <30 min Two or fewer timed steps Was one or more of the last 10 tests you performed invalid (no control line)? If YES, how many? Are there mixing wells (risk of blood splash)? Retractable needle for finger prick? Is the RDT in a cassette format (unexposed strip)? Have waste disposal safety concerns been addressed? (If no, please describe) No NA Problems /Comments

If no, what is not included:

If no, number of tests in the box affected:

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Annexes 71

Safety

Figure AS2.1 illustrates examples of RDT observations/anomalies encountered and routinely recorded during Round 5 of WHO Malaria RDT Product Testing at the CDC. In most cases, these anomalies do not invalidate the results, as reactivity in the control and test line areas are still visible, but they may pose challenges to health workers interpreting the results. Furthermore, they should be reported to manufacturers. An expanded list of notable observations concerning RDT packaging, kit accessories (buffer vials, desiccants) and instructions for use, is under development for use in both product testing and lot testing activities of the WHO-FIND Malaria RDT Evaluation Programme. Figure AS2.1: Malaria RDT anomalies encountered in production lots

a) Observations on the test strip Red background C T1 T2

Background staining is relatively common. In this example, the result is positive as test lines are positive; however, a more intense red background may obscure weak positive test lines, giving false-negative results Poor clearing of blood may obscure weak positive test lines, giving false-negative results. In this example, the result is positive as the test line is visible T2

Incomplete clearing

C

T

C

T1

b) Observations of flow problems Failure to flow C T

Blood and buffer did not run the length of the strip

Irregular migration that obscures test line(s)

C T1 T2

One portion of the nitrocellulose near the test band was not absorptive and remained dry during wicking, creating irregular migration of blood/buffer with red background that may obscure test line. One portion of the nitrocellulose near the test band was not absorptive and remained dry during wicking, creating irregular migration of blood/buffer with red background. In this example, the result is positive, as the test line is clearly visible.

Irregular migration

C

T1

72

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

c) Observations on test lines Ghost test lines C T1 T2 T3

White lines on a stained background. In this example, the result is negative, as the test line is not dark and is thus not visible. The test line is visible but interrupted (broken).

Patchy broken test line(s)

C

T1 T2

Diffuse test line(s) C T

Test line wider than control, without clearly defined edge.

d) RDT structural problems Strip misplaced in the cassette Strip can be seen only partially in the results window.



Specimen pad not seen in sample window

C T1 T2

Normally, the colour of the conjugated antibody can be seen in the sample window (commonly purple, pink or blue).

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Annexes 73

Annex S3: Selection of an appropriate RDT Figure AS3.1: How to select of an appropriate RDT

Step 1.1 Define setting of use

What? target parasite species and antigena Pf-only or mixed Pf/non-Pf infections: - HRP2 - pLDH-Pf; pLDH-pan Pf and non-Pf infections (single species)b: - HRP2, aldolase; HRP2, pLDH-pan - HRP2, pLDH-Pv; HRP2, pLDH-Pvom - HRP2, pLDH-pan; pLDH-Pv - pLDH-Pf, pLDH-pan; pLDH-Pf, pLDH-Pv - pLDH-Pf, pLDH-Pvom

P. vivax, only: - aldolase - pLDH-pan - pLDH-Pv

**Pf without HRP2 – Do not use HRP2-based RDTsc

Where?

Exposure to high temperature e.g. tropical environment OR temperature-controlled environment, including during transport and storage Laboratory personnel OR health workers outside laboratories

Who?

Step 1.2 Review RDT performance

WHO RDT product testing resultsd and apply WHO recommended RDT selection criteriae - Panel detection score - False-positivity rate - Invalid rate - Ease-of-use - Thermal stability Sensitivity and specificity based on high-quality field studies in relevant populations

Generate short-list of RDTs

Step 1.3 Apply national guidelines and experience in use of RDTs

National malaria treatment guidelines In-country experience, ease-of-use assessments (Annex 5), availability of training materials - Price - Manufacturer: production capacity, lead times, heat stability data - Delivery schedules (e.g. staggered deliveries), box size, shelf life - Registration requirements of national regulatory authorities - Product lot testing results - Overall budget requirements (Annex 5)

Step 1.4 Other considerations

or mixed Pf/non-Pf infections: Most areas of sub-Saharan Africa and lowland Papua New Guinea; Pf and non-Pf infections (single species): Most endemic areas of Asia and the Americas and isolated areas of the Horn of Africa; Mainly P. vivax-only: areas of East Asia, central Asia, South America, and some highland areas elsewhere b Tests with a P. falciparum-specific line and pan-specific line will not distinguish P. falciparum-only infections from mixed P. falciparum infections. Distinguishing P. falciparum from mixed P. falciparum-vivax infections is important only if a full course of primaquine is routinely given for infections due to P. vivax. This must be weighed against the loss of ability to detect P. malariae and P. ovale if a test has only P. falciparum- and P. vivax-specific lines. Inclusion of further test lines (e.g. Pf-Pv-pan-pLDH) to detect these increases the complexity of test interpretation. A programme should prioritize these various advantages and disadvantages according to local conditions in the initial stage of making procurement decisions. c P. falciparum parasites lacking HRP2 +/- HRP3 genes have been identified with high frequency in parts of South America (10). d See references (3–6). e WHO RDT procurement criteria : http://www.who.int/malaria/publications/atoz/rdt_selection_criteria/en/ (accessed 26 June 2014).

a Pf-only

For a comprehensive guide to procurement of malaria RDTs extending beyond selection to quantification, budgeting, technical specifications, management of tenders, contracts, supply management and monitoring of supplier performance and managing product variations, see reference (13).

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annex 1: Characteristics of RDTs evaluated in round 5 Sequence and type of bound antibodyb C T1 T2

Manufacturer G0111 F,P F,P F,P F,P F,V,O,M F,P F,V,O,M F,P F F,V F,P F,P F,P F,V F,P F,V F,P F,P F,P F,P F,P F F,P F,P P F F,P F,P F,P HRP2 pan(pLDH), F(pLDH) pan(pLDH), HRP2 pan(pLDH) HRP2 pan(pLDH), HRP2 pan(pLDH), HRP2 pan(pLDH), HRP2 pan(pLDH), HRP2 aldolase, HRP2 pan(pLDH), HRP2 pan(pLDH), HRP2 P P P P P P P P P P P P aldolase, HRP2 P V(pLDH), HRP2 P pan(pLDH), HRP2 P pan(pLDH) V(pLDH) aldolase pan(pLDH) HRP2 HRP2 pan(pLDH) HRP2 pan(pLDH) pan(pLDH) pan(pLDH) HRP2 pan(pLDH) HRP2 HRP2 HRP2, V(pLDH) P HRP2 pan(pLDH), HRP2 P pan(pLDH) HRP2 V(pLDH) HRP2 HRP2 HRP2 HRP2 pan(pLDH) aldolase HRP2 — F(pLDH) HRP2 — — HRP2 pan(pLDH) pan(pLDH) pan(pLDH), HRP2 P pan(pLDH) HRP2 pan(pLDH), HRP2 P HRP2 pan(pLDH) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 V(pLDH), HRP2 P V(pLDH) HRP2 5 HRP2 P HRP2 — 20 pan(pLDH), HRP2 P pan(pLDH) HRP2 5 4 6 3 2 4 4 3 4 4 3 4 5 5 3 3 5 5 5 5 3 4 4 VOM(pLDH), HRP2 P VOM(pLDH) HRP2 5 2 pan(pLDH), F(pLDH) P pan(pLDH) F(pLDH) 5 2 VOM(pLDH), HRP2 P VOM(pLDH) HRP2 5 4 30 20 20 20 15 15 30 20 30 20 15 15 10 30 30 20 15 15 20 20 20 20 20 30 30 pan(pLDH), HRP2 P pan(pLDH) HRP2 5 4 30 pan(pLDH), HRP2 P pan(pLDH) HRP2 5 4 20 30 — — — — — — 20 — 30 — — 30 30 20 — — 30 20 20 20 20 20 20 — — — HRP2 P HRP2 — 5 2 20 — pan(pLDH), HRP2 P pan(pLDH) HRP2 5 2 20 — G0141 RK MAL 001 A03-18-322 AM4650-K MFV-124 MAT-PF/ PAN-50 17012 0502_K25 523352 R0113C Z11200CE MALAG100 GM006 20-0146-01 IMPN-C52 AMAL-7025 ML03 412499 P pan(pLDH) P pan(pLDH) — 5 2 20 — B C A C C H C H C A E D C C F C E C C D D C A C C B A C D D

Product name

Plasmodium species targeted (F = P. falciparum V = P. vivax O = P. ovale M = P. malariae P = pan; major Catalogue Plasmodium number species)

Target antigena(s) C T1 T2

Required volume (μL) of whole blood Buffer drops (µL) A A A A A A A A A D A A A A A A A A A A A A A A A A A A A A

RecomMinimum Maximum Results mended time to reading Interprestorage resultsc tationd Format tempera- Shelf-life time e ture (min) (min) (type A-J) type (months) 1 - 40 °C 1 - 40 °C 1 - 40 °C 4 - 30 °C 2 - 30 °C 1 - 30 °C 4 - 30 °C 2 - 30 °C 1 - 30 °C 2 - 30 °C 4 - 30 °C 2 - 30 °C 2 - 30 °C 1 - 30 °C 2 - 26 °C 1 - 40 °C 4 - 30 °C 2 - 30 °C 1 - 30 °C 4 - 30 °C 2 - 30 °C 2 - 30 °C 2 - 30 °C 4 - 30 °C 4 - 30 °C 4 - 30 °C 4 - 30 °C 2 - 30 °C 4 - 30 °C 4 - 30 °C — — — 18 — — — 24 — — — 18 — — 18 — — — — 24 — — — 24 24 24 24 24 24 24

CareStart™ Malaria pLDH (PAN)

Access Bio. Inc.

CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo G0131

CareStart™ Malaria HRP2 (Pf)

Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Artron Laboratories Inc.

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

Malaria Pf/Pan Antigen (MAL Pf/Pan) Test Kit

ASAN Pharmaceutical Co., Ltd

ASAN Easy Test® Malaria Pf/Pan Ag

AZOG, INC.

Bhat Bio-Tech India (P) Ltd.

Bio Focus Co., Ltd.

Malaria pf-LDH/PAN-LDH Antigen Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test BioTracer™ Malaria Pf/PAN Rapid Card

BIOSYNEX

IMMUNOQUICK® MALARIA falciparum

Blue Cross Bio-Medical (Beijing) Co., Ltd. One Step Malaria P.F/P.V Test

CTK Biotech Inc.

OnSite Pf/Pan Ag Rapid Test

DIALAB GmbH

DIAQUICK Malaria P.f/Pan Cassette

GenBody Inc.

GenBody™ Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Genomix Molecular Diagnostics Pvt. Ltd. Test Device Green Cross Medical Science Corp. (Korea) Genedia® Malaria P.f/Pan Ag Rapid Test

Guangzhou Wondfo Biotech Co. Ltd.

Wondfo® One Step Malaria P.f/P.v Whole Blood Test W056-C

Hangzhou Biotest Biotech Co. Ltd.

RightSign™ Malaria P.f./Pan Rapid Test Cassette

Humasis Co., Ltd.

Humasis Malaria Pf/Pan Antigen Test

ICT INTERNATIONAL

ICT Malaria Dual Test

IMACCESS S.A.S

Vikia® Malaria Ag Pf/Pan

Advanced Quality™ Rapid Malaria Test (Pf/Pan)

InTec Products, Inc.

Advanced Quality™ One Step Malaria Pf Test IR231025 IR013025 IR016025

Advantage Mal Card

ITP11005 ITP11002TC1/ TC40 IR221025

J. Mitra & Co. Pvt. Ltd.

Advantage Malaria Pan + Pf Card

Advantage Pan Malaria Card

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Avantage P.f Malaria Card

LAB-CARE Diagnostics (India) PVT. LTD. ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25 MDMALLDH Medical Diagnostech (Pty) Ltd MD Malaria Pf/Pan(pLDH) test 001 NG-MALSARL NG Biotech, Z.A. NG-Test MALARIA Pf/Pan (pLDH) W23-001

(continued)

Annexes

75

76

Annex 1: Characteristics of RDTs evaluated in round 5 (continued) Sequence and type of bound antibodyb C T1 T2

Manufacturer F F,P F,P F F,V F F F,P F,P pan(pLDH) — HRP2 panpLDH 5 5 5 5 F F,P F,P e

Product name HRP2 pan(pLDH), HRP2 pan(pLDH), HRP2 HRP2 V(pLDH), HRP2 HRP2 HRP2 pan(pLDH), HRP2 pan(pLDH), HRP2 HRP2 pan(pLDH), HRP2 pan(pLDH), HRP2 P HRP2 P pan(pLDH) P HRP2 P HRP2 P pan(pLDH) HRP2 5 P HRP2 — 5 4 4 5 5 2 3 P HRP2 — 5 2 P V(pLDH) HRP2 8 4 25 20 15 15 25 20 20 20 P HRP2 — 5 3 15 P pan(pLDH) HRP2 5 4 30 P pan(pLDH) HRP2 5 2 20 — 30 30 30 — 30 — — — — — P HRP2 — 5 2 20 — A C C A E A A C D A C D

Plasmodium species targeted (F = P. falciparum V = P. vivax O = P. ovale M = P. malariae P = pan; major Catalogue Plasmodium number species)

Target antigena(s) C T1 T2

Required volume (μL) of whole blood Buffer drops (µL)

RecomMinimum Maximum Results mended time to reading Interprestorage resultsc time tationd Format tempera- Shelf-life e (min) (min) (type A-J) type ture (months) A A A A A A A A A A A A 1 - 30 °C 1 - 30 °C 1 - 40 °C 4 - 30 °C 4 - 40 °C 4 - 45 °C 1 - 40 °C 1 - 40 °C 4 - 40 °C 4 - 40 °C 4 - 30 °C 2 - 30 °C 24 24 — 24 24 24 24 24 — — 24 —

Premier Medical Corporation

RapiGEN INC. KH-R-06-20 55IC402-50 KMFC6001 05FK50/05FK53 05FK60 VB20 VB01 50310025 RT 20222

First Response® Malaria Ag P. falciparum (HRP2) I13FRC Card Test First Response® Malaria Ag. pLDH/HRP2 Combo I16FRC Card Test BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) C30RHA25

Shanghai Kehua Bio-engineering Co.,Ltd. KHB® Malaria Ag P.f Rapid Test ParaHIT®fV Rapid test for P. falciparum and Span Diagnostics Ltd. P. vivax Malaria - Device SSA Diagnostics & Biotech Systems diagnosticks-Malaria (Pf) Rapid Diagnostic Test

Standard Diagnostics Inc.

SD BIOLINE Malaria Antigen Pf

SD BIOLINE Malaria Antigen Pf/Pan

Orgenics Ltd.(IS)

Clearview® Malaria Dual

Vision Biotech (Pty) Ltd

Vision Malaria Pf

Zephyr Biomedicals

Parascreen® Rapid test for malaria Pan/Pf

Zhejiang Orient Gene Biotech Co., Ltd. Malaria Pf/Pan One Step Rapid Test

a

pLDH, plasmodium lactate dehydrogrenase; HRP2, histidine rich protein 2 b Sequence when test held in a horizontal position and the sample well is at the far right and control line, far left c From placement of buffer, or from ‘intermediate’ step, if applicable d See Annex 2

Formats include: cassette (A); card (B); hybrid (C), dipstick (D); or other (E). Each product should ideally be accompanied by all required materials (lancet, pipette, etc.) particularly when used by the village health worker level; however, this is often not the case and the contents depend on the request of the procuring agent.

A Cassette C T1 T2

B Card

C Cassette hybrid

D Dipstick

E Other

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

C

T

S

A

Pf P C

  Sample and mixing wells

C

C

T

S

T1 T2

Annex 2: Malaria RDTs: guide to interpretation of results Type A: Guide to results of generic Pf malaria RDTs Results window: C=control line; T=test line with bound HRP2 or Pf-specific pLDH antibody.

C

T

Negative results: One line ‘C’ appears in the results window. C T

Positive results:  P. falciparum infection. Two lines ‘C’ and ‘T’ appear in the results window. Test is positive even if the test line is faint. C T

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T C T

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Annexes 77

Type B: Guide to results of generic major Plasmodium species (pan) malaria RDTs Results window: C=control line; T=test line with bound pan-specific pLDH or aldolase antibody.

C

T

Negative results: One line ‘C’ appears in the results window. C T

Positive results:  Plasmodium species (P. falciparum, P. vivax, P. malariae, P. ovale) infection. Two lines ‘C’ and ‘T’ appear in the results window. Test is positive even is the test line is faint. C T

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T C T

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Type C: Guide to results of generic pan-Pf malaria RDTs Results window:  C=control line; T1=test line with bound pLDH or aldolase antibody; T2=test line with bound HRP2 and/or Pf-specific pLDH antibody.

C

T1 T2

Negative results: Only one line ‘C’ appears in the results window. C T1 T2

Positive results: P. falciparum: Two lines ‘C’ and ‘T2” appear in the results window. C T1 T2

 Non-falciparum infection (P. vivax, P. ovale, P. malariae) or mixed infection: Two lines ‘C’ and ‘T1” appear in the results window. C T1 T2

P. falciparum or mixed infection. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 C T1 T2

C T1 T2 C T1 T2

Annexes Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) 79

Type D: Guide to results of generic Pf-pan malaria RDTs Results window:  C=control line; T1=test line with bound HRP2 or Pf-specific LDH antibody; T2=test line with bound pLDH or aldolase antibody.

C

T1 T2

Negative results: Only one line ‘C’ appears in the results window. C T1 T2

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2

Non-falciparum infection (P. vivax, P. ovale, P. malariae) or mixed infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2

P. falciparum or mixed infection. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 C T1 T2

C T1 T2 C T1 T2

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Type E: Guide to results of generic Pv-Pf malaria RDTs Results window:  C=control line; T1=test line with bound P. vivax-specific pLDH; T2=test line with bound HRP2 or Pf-specific pLDH antibody.

C

T1 T2

Negative results: Only one line ‘C’ appears in the results window. C T1 T2

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2

P. vivax infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2

P. falciparum and P. vivax mixed infection. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 C T1 T2

C T1 T2 C T1 T2

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Annexes 81

Type F: Guide to results of generic Pf-Pv malaria RDTs Results window:  C=control line; T1= test line with bound HRP2 or Pf-specific pLDH antibody; T2=test line with bound P. vivax-specific pLDH.

C

T1 T2

Negative results: Only one line ‘C’ appears in the results window. C T1 T2

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2

P. vivax infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2

P. falciparum and P. vivax mixed infection. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 C T1 T2

C T1 T2 C T1 T2

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Type G: Guide to results of generic pan-Pv-Pf malaria RDTs Results window:  C=control line; T1=test line with bound pLDH or aldolase antibody; T2=test line with bound P. vivax-specific pLDH; T3=test line with bound HRP2 or Pf-specific pLDH antibody

C

T1 T2 T3

Negative results: Only one line ‘C’ appears in the results window. C T1 T2 T3

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T3’ appear in the results window. C T1 T2 T3

P. vivax infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2 T3

P. falciparum with or without mixed infection with P. ovale or P. malariae. Three lines ‘C’, ‘T1’ and ‘T3’ appear in the results window. C T1 T2 T3

P. falciparum and P. vivax mixed infection. Three lines ‘C’, ‘T2’ and ‘T3’ appear in the results window. C T1 T2 T3

P. falciparum and P. vivax mixed infection with or without P. ovale and/or P. malariae infection. Four lines ‘C’, ‘T1’, ‘T2’ and ‘T3’ appear in the results window. C T1 T2 T3

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Annexes 83

 P. vivax with or without P. ovale and/or P. malariae infection. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2 T3

P. malariae with or without P. ovale and/or P. vivax infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2 T3

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

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Type H: Guide to results of generic vom1-Pf malaria RDTs Results window:  C=control line; T1= test line with bound pLDH specific for non-P. falciparum (P. vivax, P. ovale and P. malariae); T2=test line with bound HRP2 or Pf-specific pLDH antibody

C

T1 T2

Negative results: Only one line ‘C’ appears in the results window. C T1 T2

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2

P. falciparum mixed infection (with P. vivax, P. ovale and/or P. malariae). Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2

Non-P. falciparum infection (P. vivax, P. ovale and P. malariae) or mixed infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 C T1 T2

C T1 T2 C T1 T2

1

vom, P. vivax, P. ovale, P. malariae

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annexes 85

Type I: Guide to results of generic Pv malaria RDTs Results window: C=control line; T=test line with bound P. vivax-specific pLDH.

C

T

Negative results: Only one line ‘C’ appears in the results window. C T

Positive results:  P. vivax infection. Two lines ‘C’ and ‘T’ appear in the results window. C T

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T C T

86

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Type J: Guide to results of generic Pf-Pf malaria RDTs Results window:  C=control line; T1= test line with bound pLDH specific for P. falciparum; T2=test line with bound HRP2.

C

T1 T2

Negative results: Only one line ‘C’ appears in the results window. C T1 T2

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2

P. falciparum infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2

P. falciparum infection. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 C T1 T2

C T1 T2 C T1 T2

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annexes 87

Type K: Guide to results of generic Pv-Pf-Pf malaria RDTs Results window:  C=control line; T1= test line with bound P. vivax-specific pLDH; T2=test line with bound HRP2 or Pf-specific pLDH antibody; T3=test line with bound HRP2 or Pf-specific pLDH antibody. If an RDT has bound HRP2 antibodies on T2, T3 will have bound Pf-specific pLDH and vice versa (T2 Pf antigen target ≠ T3 Pf antigen target).

C

T1 T2 T3

Negative results: Only one line ‘C’ appears in the results window. C T1 T2 T3

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T3’ appear in the results window. C T1 T2 T3

P. falciparum infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2 T3

P. falciparum infection. Three lines ‘C’, ‘T2’ and ‘T3’ appear in the results window. C T1 T2 T3

P. falciparum infection and P. vivax mixed infection. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2 T3

P. falciparum infection and P. vivax mixed infection. Three lines ‘C’, ‘T1’ and ‘T3’ appear in the results window. C T1 T2 T3

88

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

 P. falciparum infection and P. vivax mixed infection. Four lines ‘C’, ‘T1’, ‘T2’ and ‘T3’ appear in the results window. C T1 T2 T3

P. vivax infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2 T3

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annexes 89

Type L: Guide to results of generic pan-Pf-Pf malaria RDTs Results window:  C=control line; T1= test line with bound PAN-pLDH or aldolase antibody; T2=test line with bound HRP2 or Pf-specific pLDH antibody; T3=test line with bound HRP2 or Pf-specific pLDH antibody. If an RDT has bound HRP2 antibodies on T2, T3 will have bound Pf-specific pLDH and vice versa (T2 Pf antigen target ≠ T3 Pf antigen target)

C

T1 T2 T3

Negative results: Only one line ‘C’ appears in the results window. C T1 T2 T3

Positive results: P. falciparum infection. Two lines ‘C’ and ‘T2’ appear in the results window. C T1 T2 T3

P. falciparum infection. Two lines ‘C’ and ‘T3’ appear in the results window. C T1 T2 T3

P. falciparum infection. Three lines ‘C’, ‘T2’ and ‘T3’ appear in the results window. C T1 T2 T3

P. falciparum infection with or without mixed infection with P. vivax, P. ovale and/or P. malariae. Three lines ‘C’, ‘T1’ and ‘T2’ appear in the results window. C T1 T2 T3

P. falciparum infection with or without mixed infection with P. vivax, P. ovale and/or P. malariae. Three lines ‘C’, ‘T1’ and ‘T3’ appear in the results window. C T1 T2 T3

90

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

 P. falciparum infection with or without mixed infection with P. vivax, P. ovale and/or P. malariae. Four lines ‘C’, ‘T1’, ‘T2’ and ‘T3’ appear in the results window. C T1 T2 T3

Non-P. falciparum infection (P. vivax, P. ovale, P. malariae) or mixed infection. Two lines ‘C’ and ‘T1’ appear in the results window. C T1 T2 T3

Invalid results:  No ‘C’ line appears in the results window. Repeat the test with a new RDT if no control line appears. C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

C T1 T2 T3 C T1 T2 T3

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annexes 91

92

Annex 3: Phase-1 results P. falciparum samples (n=20) Total positive results returned Manufacturer Lot 1 Test 1 Test 2 No. positive agreementsb (max=20) Test 1 Test 2 19.0 19.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 17.0 (19) 20.0 7.0 18.0 19.0 20.0 20.0 20.0 19.0 20.0 19.0 20.0 20.0 20.0 20.0 17.0 20.0 12.0 20.0 20.0 19.0 20.0 19.0 19.0 20.0 19.0 16.0 19.0 (19) 13.0 20.0 20.0 19.0 19.0 19.0 19.0 20.0 19.0 15.0 19.0 (19) 7.0 20.0 20.0 20.0 20.0 7.0 20.0 20.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 18.0 20.0 20.0 15.0 19.0 (19) 15.0 20.0 19.0 (19) 20.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 19.0 18.0 20.0 20.0 20.0 19.0 19.0 20.0 20.0 18.0 (19) 19.0 4.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 19.0 19.0 19.0 20.0 20.0 13.0 19.0 (19) 7.0 20.0 20.0

TableA3.1: Lot variability in positive resultsa against phase-1 P. falciparum culture samples at low (200) and high (2000) parasite density (parasites/μL)

Product Lot 2

Catalogue number

200 parasites/μL

2000 parasites/μL Lot 1 No. positive agreementsb (max=20) 19.0 18.0 20.0 20.0 20.0 19.0 19.0 20.0 20.0 18.0 (19) 19.0 0.0 20.0 20.0 20.0 20.0 20.0 20.0 19.0 19.0 19.0 18.0 20.0 20.0 12.0 18.0 (18) 4.0 20.0 19.0 (19)

Lot 2 Test 1 Test 2

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 20.0 20.0 12.0 20.0 19.0 20.0 20.0 20.0 19.0 17.0 (19) 20.0 9.0 18.0 20.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 19.0 19.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 19.0 (19) 20.0 20.0 20.0 19.0 (19) 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 20.0 20.0 LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A.

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

20.0 20.0 20.0 20.0 20.0 20.0 19.0 (19) 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0

Pf only Advanced Quality™ One Step Malaria Pf Test Avantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria Pf/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH)

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001

Table A3.1 (continued) P. falciparum samples (n=20) Total positive results returned Manufacturer Lot 1 Test 1 CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. 19.0 19.0 16.0 20.0 15.0 19.0 18.0 20.0 20.0 19.0 20.0 20.0 19.0 20.0 20.0 18.0 (19) 19.0 19.0 20.0 20.0 20.0 18.0 (19) 19.0 19.0 19.0 20.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 18.0 (19) 20.0 20.0 19.0 19.0 18.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 19.0 20.0 19.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 19.0 20.0 18.0 (19) 20.0 20.0 19.0 19.0 17.0 20.0

Product Lot 2 No. positive agreementsb (max=20) Test 1 Test 2 No. positive agreementsb (max=20) Test 1 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0

Catalogue number Lot 1 Test 2

200 parasites/μL

2000 parasites/μL Lot 2 Test 2 20.0 20.0 20.0 20.0 20.0 20.0 19.0 20.0 20.0 20.0 20.0 20.0 20.0

R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a Results are based on the first reader’s interpretation according to the manufacturer’s instructions. b Number of samples that returned a positive result for both tests. Where one test was invalid and the other positive, positive agreement was recorded.

Annexes

93

94

Table A3.2: Distribution of test band intensity (0-4) scores against phase-1 P. falciparum cultured parasites at low (200) and high (2000) parasite density (parasites/μL) 200 parasites/μL Manufacturer 0a 1 57.5 17.5 7.5 5.0 6.3 28.8 12.5 3.8 10.0 60.0 41.3 33.8 33.8 57.5 45.0 22.5 46.3 16.3 42.5 45.0 43.8 8.8 30.0 63.8 35.0 2.5 8.8 16.3 16.3 0.0 11.3 10.0 8.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 2.5 0.0 0.0 15.0 2.5 0.0 2.5 25.0 5.0 5.0 5.0 65.0 7.5 5.0 7.5 70.0 92.5 95.0 95.0 20.0 90.0 95.0 90.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 33.8 5.0 0.0 0.0 2.5 27.5 42.5 27.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA

2000 parasites/μL Percentage distribution of Pf test band intensityb (n=40) 0a 1 2 3 4 1 2 3 4 0a 0a 1 NA NA NA NA NA NA NA NA NA 40.0 32.5 72.5 0.0 12.5 62.5 0.0 45.0 10.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0 40.0 0.0 5.0 2.5 2.5 2.5 2.5 0.0 10.0 2.5 60.0 0.0 27.5 12.5 20.0 5.0 17.5 5.0 40.0 17.5 0.0 2.5 67.5 85.0 77.5 92.5 80.0 92.5 47.5 80.0 0.0 97.5 82.5 77.5 76.3 97.5 82.5 70.0 26.3 67.5 76.3 22.5 17.5 20.0 23.8 1.3 17.5 28.8 42.5 32.5 21.3 70.0 0.0 2.5 0.0 1.3 0.0 1.3 28.8 0.0 2.5 7.5 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 52.5 0.0 0.0 0.0 0.0 45.0 0.0 60.0 60.0 55.0 35.0 37.5 27.5 2.5 7.5 52.5 0.0

200 parasites/μL Percentage distribution of pan test band intensityb (n=80)

2000 parasites/μL Percentage distribution of pan test band intensityb (n=40) 2 NA NA NA NA NA NA NA NA NA 55.0 57.5 27.5 52.5 70.0 37.5 67.5 47.5 87.5 40.0 40.0 40.0 12.5 62.5 65.0 45.0 90.0 2.5 60.0

Product 2 3 4

Catalogue number

Percentage distribution of Pf test band intensityb (n=80)

3 NA NA NA NA NA NA NA NA NA 0.0 7.5 0.0 47.5 17.5 0.0 32.5 2.5 2.5 0.0 0.0 2.5 0.0 0.0 5.0 45.0 2.5 0.0 37.5

4 NA NA NA NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 7.5 0.0 0.0 2.5

Pf only InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 3.8 0.0 0.0 0.0 5.0 1.3 0.0 0.0 3.8

ITP11002TC1/ TC40 Avantage P.f Malaria Card IR016025 CareStart™Malaria HRP2 (Pf) G0141 diagnosticks- Malaria (Pf)Cassette WB KMFC6001 First Response® Malaria Ag P. falciparum (HRP2) Card Test I13FRC 0502_K25 IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test KH-R-06-20 05FK50/05FK53 SD BIOLINE Malaria Antigen Pf Vision Malaria Pf VB01 Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25 Advanced Quality™ Rapid Malaria Test (Pf/Pan) ITP11005 Advantage Mal Card IR221025 Advantage Malaria Pan + Pf Card IR231025 BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) C30RHA25 BioTracer™ Malaria Pf/PAN Rapid Card 17012 CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo G0131 6.3 1.3 60.0 2.5 1.3 0.0 0.0 0.0 1.3 2.5 2.5 2.5 5.0 0.0 1.3 23.8 3.8 41.3 0.0 0.0 0.0 0.0 1.3 0.0 2.5 3.8 11.3 5.0 42.5 2.5 27.5 31.3 58.8 43.8 47.5 27.5 52.5 48.8 27.5 42.5 8.8 56.3 16.3 16.3 2.5 8.8 0.0 13.8 1.3 27.5 11.3 15.0 8.8 6.3 28.8 5.0 57.5 1.3 46.3 46.3 51.3 53.8 28.8 35.0 50.0 1.3 30.0 16.3 32.5 23.8 33.8 42.5 11.3 31.3 0.0 55.0 7.5 7.5 5.0 3.8 21.3 1.3 10.0 0.0 13.8 26.3 56.3 11.3 3.8 0.0 15.0 26.3 40.0 32.5 7.5 23.8 8.8 6.3 7.5 58.8 43.8 0.0 57.5 38.8 51.3 42.5 30.0 61.3 16.3 25.0 0.0 7.5 47.5 25.0 37.5 51.3 26.3 3.8 3.8 0.0 0.0 5.0 0.0 11.3 12.5 3.8 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 25.0 0.0 0.0 0.0 0.0 0.0 0.0 7.5 7.5 75.0 5.0 2.5 5.0 2.5 0.0 0.0 32.5 22.5 0.0 32.5 7.5 17.5 5.0 5.0 20.0 60.0 67.5 0.0 62.5 90.0 77.5 92.5 95.0 80.0 52.5 58.8 95.0 10.0 35.0 65.0 12.5 38.8 32.5 42.5 41.3 5.0 87.5 53.8 35.0 85.0 57.5 65.0 5.0 0.0 0.0 2.5 11.3 0.0 2.5 1.3 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.0

Advanced Quality™ One Step Malaria Pf Test

Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

VB20 Z11200CE

5.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 2.5 5.0 0.0 10.0 10.0 27.5 2.5 50.0 5.0 27.5 90.0 67.5 95.0 45.0 95.0 62.5 18.8 83.8 91.3 78.8 57.5 100.0 77.5 16.3 7.5 21.3 42.5 0.0 2.5 0.0 1.3 0.0 0.0 0.0 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.0 20.0 0.0 10.0 2.5 30.0 62.5 80.0 5.0 87.5 55.0 65.0 32.5 0.0 60.0 2.5

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria Pf/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test

NG-Test MALARIA Pf/Pan (pLDH)

40.0 5.0 0.0 0.0 35.0 0.0

2.5 0.0 0.0 0.0 0.0 0.0

OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MALW23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

Table A3.2 (continued) 200 parasites/μL Manufacturer 0a 1 13.8 3.8 16.3 1.3 42.5 8.8 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 11.3 1.3 85.0 95.0 3.8 3.8 0.0 0.0 42.5 41.3 6.3 0.0 0.0 2.5 10.0 87.5 NA NA NA NA 47.5 6.3 0.0 0.0 0.0 10.0 45.0 45.0 NA NA NA NA NA NA 0.0 0.0 NA NA 0.0 0.0 50.0 43.8 66.3 40.0 28.8 40.0 15.0 48.8 7.5 6.3 1.3 8.8 0.0 2.5 0.0 0.0 0.0 0.0 0.0 0.0 5.0 2.5 0.0 2.5 15.0 0.0 60.0 5.0 80.0 95.0 40.0 92.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0

2000 parasites/μL Percentage distribution of Pf test band intensityb (n=40) 0a 1 2 3 4 1 2 3 4 1 2 NA NA NA NA NA NA 50.0 37.5

200 parasites/μL Percentage distribution of pan test band intensityb (n=80) 0a 0a 3 NA NA NA NA NA NA 47.5 55.0

2000 parasites/μL Percentage distribution of pan test band intensityb (n=40) 4 NA NA NA NA NA NA 2.5 7.5

Product 2 3 4

Catalogue number

Percentage distribution of Pf test band intensityb (n=80)

AM4650-K GM006 MAT-PF/PAN-50 523352 Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. Pvom, Plasmodium vivax, ovale and malariae NA NA 1.3 3.8

ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd.

0.0 6.3 1.3 1.3

55IC402-50

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  a Denotes no band visible b Calculations include invalid tests

pan, Plasmodium species 

Annexes

95

96

Annex 4: Phase-2 results P. falciparum samples (n=100) Total positive resultsa returned 200 parasites/μL Lot 1 Test 1 No. positive Test 2 agreementsc Test 1 (max=100) Test 2 Test 1 63.0 92.0 93.0 91.0 (99) 97.0 75.0 83.0 96.0 89.0 72.0 81.0 (90) 42.0 87.0 79.0 82.0 91.0 93.0 91.0 84.0 87.0 85.0 70.0 92.0 (99) 98.0 65.0 (99) 92.0 91.0 (98) 88.0 (98) 89.0 90.0 94.0 78.0 94.0 96.0 96.0 97.0 91.0 94.0 86.0 88.0 90.0 79.0 91.0 (98) 93.0 (99) 97.0 96.0 68.0 66.0 (99) 97.0 96.0 94.0 90.0 93.0 85.0 86.0 89.0 77.0 88.0 (97) 94.0 64.0 (99) 95.0 96.0 (99) 43.0 90.0 81.0 87.0 98.0 44.0 91.0 82.0 86.0 95.0 (99) 35.0 87.0 79.0 82.0 80.0 79.0 75.0 97.0 99.0 95.0 98.0 92.0 97.0 90.0 100.0 100.0 99.0 100.0 97.0 92.0 91.0 89.0 (95) 95.0 (97) 89.0 (99) 86.0 84.0 (99) 93.0 100.0 NA NA NA NA 94.0 (99) 23.0 (34) 97.0 (97) 100.0 96.0 95.0 100.0 100.0 98.0 (98) 99.0 97.0 97.0 100.0 100.0 100.0 100.0 100.0 100.0 98.0 100.0 98.0 98.0 100.0 100.0 99.0 100.0 100.0 100.0 100.0 98.0 99.0 (99) 100.0 94.0 (99) 97.0 96.0 95.0 100.0 100.0 NA NA NA NA NA NA 27.0 28.0 (34) 23.0 (31) 35.0 35.0 35.0 35.0 33.0 31.0 32.0 34.0 34.0 26.0 33.0 32.0 (34) 32.0 29.0 14.0 35.0 26.0 8.0 35.0 30.0 33.0 34.0 31.0 24.0 10.0 33.0 (34) 23.0 7.0 99.0 (99) 34.0 (34) 33.0 (34) 94.0 96.0 95.0 94.0 98.0 94.0 93.0 95.0 93.0 99.0 100.0 100.0 100.0 99.0 (99) 99.0 (99) NA NA NA NA NA NA NA NA NA NA NA NA NA NA 17.0 (34) 21.0 (30) 35.0 35.0 30.0 31.0 34.0 24.0 31.0 31.0 (34) 29.0 21.0 8.0 33.0 (34) 17.0 4.0 32.0 (33) 59.0 (99) 85.0 83.0 NA 73.0 93.0 99.0 (99) NA NA NA NA NA NA NA NA NA NA NA 29.0 34.0 35.0 35.0 32.0 32.0 35.0 28.0 35.0 33.0 32.0 27.0 10.0 35.0 29.0 2.0 35.0

Table A4.1: Lot variation in positive results against phase-2 wild-type P. falciparum and P. vivax samples at low (200) and high (2000) parasite density (parasites/μL) P. vivax samples (n=35) Total positive resultsa returned 2000b parasites/μL 200 parasites/μL Lot 1 No. positive Test 2 agreementsc Test 1 (max=35) Lot 2 Lot 2 Lot 2 Lot 1 No. positive c Test 2 agreements Test 1 (max=100)

Product

Catalogue number

Manufacturer

2000b parasites/μL Lot 2 Test 2 Lot 1 No. positive c Test 2 agreements Test 1 (max=35) NA NA NA NA NA NA NA NA NA 24.0 31.0 (34) 33.0 35.0 29.0 33.0 34.0 32.0 33.0 31.0 34.0 28.0 12.0 32.0 (34) 31.0 3.0 33.0 NA NA NA NA NA NA NA NA NA 23.0 31.0 (34) 33.0 35.0 29.0 30.0 34.0 27.0 33.0 29.0 31.0 23.0 6.0 32.0 (34) 26.0 2.0 33.0 NA NA NA NA NA NA NA NA NA 35.0 34.0 34.0 35.0 34.0 35.0

Pf only 70.0 (99) NA NA NA NA NA NA NA NA NA 34.0 35.0 35.0 34.0 (34) 35.0 35.0 35.0 35.0 32.0 33.0 34.0(34) 32.0(33) 35.0 35.0 35.0 33.0 34.0 33.0 35.0 34.0 (34) 35.0 35.0 31.0 32.0 35.0 35.0 34.0 35.0

Advanced Quality™ One Step Malaria Pf Test

InTec Products, Inc.

ITP11002TC1/ TC40 IR016025 G0141 KMFC6001 97.0 82.0 90.0 96.0 92.0 79.0 91.0 (96) 87.0 (94) 48.0 50.0 91.0 92.0 82.0 82.0 84.0 87.0 94.0 95.0 93.0 85.0 89.0 87.0 76.0 95.0 (99) 99.0 74.0 (99) 94.0 99.0 69.0 72.0 91.0 91.0 87.0 93.0 95.0 94.0 77.0 96.0 93.0 84.0 81.0 99.0

J. Mitra & Co. Pvt. Ltd. 92.0 95.0 Access Bio. Inc. 96.0 94.0 SSA Diagnostics & Biotech Systems 93.0 (99) 96.0 (100)

Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) I13FRC Card Test 0502_K25 IMMUNOQUICK® MALARIA falciparum

Premier Medical Corporation

KHB® Malaria Ag P.f Rapid Test

SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan

BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. 05FK50/05FK53 Standard Diagnostics Inc. VB01 Vision Biotech (Pty) Ltd

KH-R-06-20

ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test MAGC 25

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card

ITP11005 IR221025 IR231025 C30RHA25 17012

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd.

CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo G0131 Clearview® Malaria Dual VB20 DIAQUICK Malaria P.f/Pan Cassette Z11200CE

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit RK MAL 001

Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. )

First Response® Malaria Ag. pLDH/HRP2 Combo I16FRC Card Test GenBody™Malaria Pf/Pan Ag MALAG100

Premier Medical Corporation

Genedia® Malaria P.f/Pan Ag Rapid Test

20-0146-01

GenBody Inc. Green Cross Medical Science Corp. (Korea)

Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit

AMAL-7025 ML03 A03-18-322

Malaria Pf/Pan One Step Rapid Test

RT 20222

Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd.

Table A4.1 (continued) P. falciparum samples (n=100) Total positive resultsa returned 200 parasites/μL Lot 1 Test 1 76.0 98.0 94.0 100.0 100.0 100.0 99.0 (99) 100.0 94.0 100.0 100.0 97.0 (99) 100.0 93.0 (96) 99.0 (99) 99.0 (99) 100.0 100.0 91.0 100.0 98.0 99.0 100.0 98.0 98.0 99.0 100.0 27.0 (34) 35.0 23.0 28.0 35.0 35.0 32.0 99.0 29.0 28.0 34.0 29.0 35.0 23.0 16.0 35.0 35.0 99.0 8.0 (34) 10.0 99.0 33.0 34.0 32.0 5.0 (34) 24.0 31.0 25.0 (34) 35.0 20.0 14.0 35.0 35.0 35.0 6.0 23.0 30.0 (33) 30.0 35.0 24.0 26.0 35.0 35.0 34.0 24.0 24.0 35.0 24.0 21.0 34.0 19.0 17.0 33.0 28.0 25.0 90.0 95.0 84.0 97.0 89.0 89.0 92.0 94.0 95.0 73.0 90.0 82.0 91.0 82.0 85.0 87.0 96.0 80.0 93.0 78.0 92.0 89.0 94.0 94.0 (99) 90.0 (99) 63.0 (99) 83.0 81.0 80.0 95.0 98.0 97.0 96.0 86.0 (99) 95.0 98.0 94.0 85.0 (94) 92.0 (99) 94.0 (98) 90.0 (97) 86.0 87.0 86.0 84.0 89.0 86.0 91.0 88.0 87.0 96.0 95.0 98.0 96.0 95.0 88.0 (99) 95.0 78.0 88.0 (99) 94.0 76.0 92.0 96.0 93.0 91.0 98.0 88.0 88.0 96.0 87.0 95.0 93.0 97.0 94.0 (99) 93.0 (99) 100.0 33.0 35.0 33.0 31.0 25.0 34.0 24.0 25.0 34.0 6.0 22.0 33.0 33.0 35.0 26.0 21.0 35.0 31.0 75.0 99.0 65.0 97.0 68.0 99.0 70.0 96.0 54.0 96.0 99.0 100.0 98.0 100.0 14.0 16.0 18.0 19.0 9.0 14.0 23.0 25.0 15.0 26.0

P. vivax samples (n=35) Total positive resultsa returned 2000b parasites/μL 200 parasites/μL Lot 1 Test 1 No. positive Test 2 agreementsc Test 1 (max=35) 11.0 19.0 23.0 32.0 22.0 21.0 34.0 3.0 17.0 28.0 (33) 29.0 35.0 20.0 17.0 35.0 31.0

Product Lot 2 Test 2 Lot 2 Lot 2 No. positive Test 2 agreementsc Test 1 (max=100) Lot 1 No. positive c Test 2 agreements Test 1 (max=100)

Catalogue number

Manufacturer

2000b parasites/μL Lot 2 Test 2 23.0 19.0 34.0 35.0 34.0 31.0 35.0 33.0 (34) 35.0 34.0 34.0 (34) 35.0 30.0 35.0 35.0 35.0 22.0 23.0 33.0 34.0 35.0 35.0 34.0 34.0 35.0 34.0 35.0 35.0 35.0 34.0 35.0 34.0

Lot 1 No. positive c Test 2 agreements Test 1 (max=35)

Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test

AZOG, INC. Medical Diagnostech (Pty) Ltd

SARL NG Biotech, Z.A.

CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd.

Standard Diagnostics Inc.

MFV-124 MDMALLDH001 NG-MALNG-Test MALARIA Pf/Pan (pLDH) W23-001 OnSite Pf/Pan Ag Rapid Test R0113C Parascreen® Rapid test for malaria Pan/Pf 50310025 RightSign™ Malaria P.f./Pan Rapid Test Cassette IMPN-C52 05FK60/ SD BIOLINE Malaria Antigen Pf/Pan 05FK63 Vikia® Malaria Ag Pf/Pan 412499 Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag AM4650-K

IMACCESS S.A.S

Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device 91.0 (99) 98.0 70.0 (99) 94.0 88.0 91.0

GM006

ASAN Pharmaceutical Co., Ltd 91.0 Genomix Molecular Diagnostics 89.0 (94) Pvt. Ltd.

Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line MAT-PF/ Antigen Test PAN-50

Bhat Bio-Tech India (P) Ltd.

One Step Malaria P.F/P.V Test (Cassette)

523352

Blue Cross Bio-Medical (Beijing) Co., Ltd.

ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device

55IC402-50

Span Diagnostics Ltd.

Wondfo® One Step Malaria P.f/P.v Whole Blood Test W056-C

Guangzhou Wondfo Biotech Co. Ltd.

Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

IR013025 G0111

J. Mitra & Co. Pvt. Ltd. Access Bio. Inc.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a Results are based on the first reader’s interpretation according to the manufacturer’s instructions. b 4 (4%) of the 100 P. falciparum dilution samples sets had 200 and 5000 parasites/μL and 2 (6%) of the 35 P. vivax dilution sample sets had 200 and 5000 parasites/μL c Number of samples that returned a positive result for both tests. Where one test was invalid and the other positive, positive agreement was recorded.

Annexes

97

98

Table A4.2: Distribution of test band intensity (0-4) scores against phase-2 wild-type P. falciparum samples at low (200) and high (2000) parasite density (parasites/μL)

Product

Catalogue number

Manufacturer

200 parasites/μL Percentage distribution of Pf test band intensityc (n=400) 0a 1 2 3 4 24.5 46.8 24.8 6.3 27.3 42.3 18.3 6.0 4.0 18.0 32.5 31.3 14.3 5.3 14.8 26.0 28.0 26.0 2.8 13.5 28.5 33.3 22.0 15.5 33.0 37.5 13.3 9.3 22.3 33.5 22.3 12.8 2.8 10.5 26.3 36.8 23.8 7.0 19.0 35.5 28.8 9.8 21.3 34.8 34.0 10.0 0.0 3.5 0.5 0.0 0.0 0.0 0.0 0.0 1.3 0.5 0.3 1.3 1.5 4.0 10.5 0.5 1.0 0.3 4.8 3.0 0.0 0.0 0.5 0.5 11.5 72.0 92.3 6.8 4.5 23.5 71.0 62.5 36.5 6.5 16.0 76.0 62.5 30.5 1.0 0.8 5.8 4.0 12.5 20.0 34.5 29.0 40.8 46.5 12.5 0.5 0.0 10.0 25.5 64.0 62.0 37.3 1.5 26.5 48.5 21.0 2.5 7.8 91.0 0.8 1.3 0.3 0.0 0.0 0.0 0.0 0.0 0.3 1.3 0.3 0.0 0.0 0.0 0.0 0.0 2.3 0.0 2.5 8.5 1.8 0.0 0.5 0.0 0.0 0.0 0.0 2.0 1.0 0.5 2.5 0.5 2.0 12.0 85.5 21.0 66.3 12.8 7.5 17.0 72.5 65.5 32.8 11.5 24.5 63.5 62.5 37.0 4.5 26.5 50.0 18.5 0.5 0.0 1.5 14.0 20.5 64.0 1.5 2.0 9.5 23.5 63.5 3.0 2.0 13.0 24.0 58.0 69.3 12.3 78.5 78.0 30.8 0.0 62.8 25.0 20.5 1.0 21.8 0.3 1.5 2.5 11.5 33.5 51.0 70.0 29.5 4.5 5.5 22.0 36.0 32.0 81.0 15.5 7.0 34.3 42.3 14.3 53.8 9.0 18.3 14.0 5.5 14.0 31.5 23.5 25.5 5.8 20.3 39.3 27.3 25.3 22.8 24.3 22.5 8.8 4.0 9.5 3.5 2.3 7.5 44.8 1.5 0.0 32.3 43.0 13.3 12.8 31.5 29.0 24.5 40.5 19.3 0.5 0.0 0.0 0.5 3.5 12.0 84.0 6.5 18.5 74.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4.0 0.5 7.5 14.0 74.0 NA NA NA NA NA NA NA NA NA NA NA 1.5 9.0 42.5 44.5 0.0 5.5 42.5 0.0 2.0 2.0 0.0 15.0 21.0 0.0 9.5 41.0 0.0 0.3 0.0 0.0 0.0 1.0 4.0 0.8 3.5 4.5 17.5 37.0 37.5 NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 47.5 4.5 0.0 42.5 43.5 10.0 51.0 12.0 1.0 49.0 0.5 0.0 8.5 49.0 33.0 5.0 50.0 45.0 0.0 4.5 24.5 57.0 14.0 7.0 33.5 51.0 3.0 36.0 53.0 0.0 15.5 43.5 39.0 0.0 43.0 28.5 28.0 0.0 0.0 0.0 0.0 0.0 8.0 7.5 2.0 0.5 3.0 29.5 56.5 10.5 4.5 23.5 48.0 23.5 8.5 18.5 39.5 31.5 2.5 28.0 58.5 6.0 89.5 4.0 11.0 0.5 8.5 0.0 0.0 0.5 0.5 0.0 0.0 0.5 0.5 2.0 0.0 0.0 0.0 0.5 2.0 15.0 82.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 1.0 0.0 2.0 10.0 87.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.5 0.0 2.5 1.0 11.0 17.0 69.0 4.0 19.0 76.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 3.8 0.3 4.0 13.0 29.0 31.0 23.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

2000b parasites/μL 200 parasites/μL 2000b parasites/μL 200 parasites/μL Percentage distribution Percentage distribution of Percentage distribution of Percentage distribution of Pf test band intensityc pan test band intensityc pan test band intensityc of Pv test band intensityc (n=200) (n=400) (n=200) (n=400) 0a 1 2 3 4 0a 1 2 3 4 0a 1 2 3 4 0a 1 2 3 4

2000b parasites/μL Percentage distribution of Pv test band intensityc (n=200) 0a 1 2 3 4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf)

ITP11002TC1/ TC40 IR016025 G0141

InTec Products, Inc.

diagnosticks- Malaria (Pf)Cassette WB KMFC6001

J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems

First Response® Malaria Ag P. falciparum I13FRC (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum 0502_K25

Premier Medical Corporation

KHB® Malaria Ag P.f Rapid Test

KH-R-06-20

BIOSYNEX Shanghai Kehua Bioengineering Co.,Ltd.

SD BIOLINE Malaria Antigen Pf

05FK50/05FK53 Standard Diagnostics Inc. Vision Biotech (Pty) Ltd

Vision Malaria Pf VB01 Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan MAGC 25 Antigen Test

LAB-CARE Diagnostics (India) PVT. LTD.

0.0 21.5 46.0 31.0

ITP11005

InTec Products, Inc.

IR221025 IR231025 C30RHA25 17012

J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd.

Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual

G0131

Access Bio. Inc.

VB20

Orgenics Ltd.(IS)

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) 2.0 13.0 19.0 66.0 74.5 25.3 7.0 14.0 78.0 69.0 29.8 2.0 12.0 26.0 60.0 86.5 12.0 23.8 21.8 26.0 19.8 6.8 17.3 38.8 30.8 6.5 2.3 17.8 35.3 28.3 16.5 4.8 5.8 18.3 43.8 27.5 27.8 69.3

DIAQUICK Malaria P.f/Pan Cassette

Z11200CE

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 I16FRC Combo Card Test GenBody™Malaria Pf/Pan Ag MALAG100

DIALAB GmbH 5.8 22.0 44.8 Advy Chemical Pvt. Ltd. (Affiliate of Bharat 13.3 21.3 39.3 Serums & Vaccines Ltd. ) Premier Medical 10.5 21.0 34.8 Corporation GenBody Inc. 9.5 26.8 37.3

Genedia® Malaria P.f/Pan Ag Rapid Test 20-0146-01

Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL

Humasis Malaria Pf/Pan Antigen Test AMAL-7025 ICT Malaria Dual Test ML03 Malaria Pf./Pan Antigen (MAL Pf/Pan) A03-18-322 Test Kit

Artron Laboratories Inc. 30.8 38.3 26.0

Malaria Pf/Pan One Step Rapid Test

RT 20222

Zhejiang Orient Gene Biotech Co., Ltd.

Malaria pf (pLDH) / PAN-pLDH Test Device MFV-124

AZOG, INC.

Table A4.2 (continued)

Product

Catalogue number

Manufacturer

200 parasites/μL Percentage distribution of Pf test band intensityc (n=400) 0a 1 2 3 4 2.0 16.3 36.0 34.3 35.5 29.3 8.5 39.8 24.8 3.0 34.0 26.8 16.8 48.0 10.5 28.3 35.5 20.3 47.3 17.8 0.8 2.3 7.0 4.0 NA NA NA NA 15.8 61.8 22.5 7.3 64.3 28.0 0.0 0.5 0.0 0.0 NA NA NA NA NA 2.0 1.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 4.5 1.0 NA NA NA NA NA NA NA NA NA NA 0.5 7.0 12.0 79.5 5.0 21.5 26.5 42.5 0.0 8.0 20.0 72.0 0.0 0.0 13.5 42.0 44.5 NA NA NA NA 4.0 1.0 9.0 20.5 65.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA 1.5 43.5 44.0 9.0 2.0 25.0 43.5 28.5 0.5 3.0 12.0 26.0 58.5 NA NA NA NA NA NA NA NA NA NA 83.5 15.5 98.5 1.3 72.8 25.8 78.5 19.0 98.0 98.5 NA NA 1.3 1.5 NA NA 0.5 2.0 0.5 5.0 13.5 80.5 36.0 53.3 10.5 3.0 12.0 32.5 50.5 82.8 15.0 2.3 0.3 0.0 0.0 0.0 2.0 9.0 46.5 33.5 11.0 52.0 34.0 3.0 9.0 0.0 NA NA NA NA NA NA 1.0 0.3 1.5 2.5 0.3 0.0 NA NA 0.8 3.0 2.5 17.5 41.5 35.5 84.8 14.8 0.0 0.5 0.0 24.5 66.5 9.0 0.0 0.0 NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA 0.0 0.0 0.5 0.0 2.5 0.0 7.0 29.0 64.0 38.0 50.3 10.5 9.5 26.5 61.5 68.3 31.0 0.5 6.0 9.0 84.5 67.3 32.5 0.3 1.3 0.3 0.0 0.0 0.0 0.0 2.5 15.5 46.5 32.0 4.0 31.0 52.5 12.0 6.5 36.0 45.0 12.0 3.5 0.5 0.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 11.5 0.0 0.0 6.0 23.5 70.5 28.3 56.8 14.3 0.8 0.0 2.5 7.5 50.0 37.0 3.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 14.5 47.0 34.5 0.0 96.5 3.0 0.5 0.0 13.0 58.5 28.0 0.0 91.0 0.5 99.5 0.0 98.5 NA NA NA NA 8.0 0.0 1.0 NA NA 1.0 0.0 0.0 NA NA NA NA NA NA NA NA NA 4.0 0.0 0.5 0.0 0.5 0.0 NA NA

2000b parasites/μL 200 parasites/μL 2000b parasites/μL 200 parasites/μL Percentage distribution Percentage distribution of Percentage distribution of Percentage distribution of Pf test band intensityc pan test band intensityc pan test band intensityc of Pv test band intensityc (n=200) (n=400) (n=200) (n=400) 0a 1 2 3 4 0a 1 2 3 4 0a 1 2 3 4 0a 1 2 3 4

2000b parasites/μL Percentage distribution of Pv test band intensityc (n=200) 0a 1 2 3 4 NA NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.5 NA NA

MD Malaria Pf/Pan(pLDH) test

MDMALLDH001

Medical Diagnostech (Pty) Ltd

NG-Test MALARIA Pf/Pan (pLDH)

OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan

Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom 11.8 28.3 37.0 20.8 8.3 20.3 38.8 25.8 5.5 22.0 46.5 22.0 3.0 17.5 41.3 27.8 10.5 23.3 26.8 38.0 7.0 15.0 35.5 27.8 14.8 NA NA NA NA NA NA NA NA NA NA 11.0 1.0

NG-MAL-W23-001 SARL NG Biotech, Z.A. 5.0 21.8 R0113C CTK Biotech Inc. 9.8 22.8 50310025 Zephyr Biomedicals 4.0 18.5 Hangzhou Biotest 14.3 26.5 IMPN-C52 Biotech Co. Ltd. 05FK60/05FK63 Standard Diagnostics Inc. 3.3 12.8 412499 IMACCESS S.A.S 10.8 23.5

ASAN Easy Test® Malaria Pf/Pan Ag

AM4650-K

Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device

GM006

ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd.

MAT-PF/PAN-50

Bhat Bio-Tech India (P) Ltd.

523352

Blue Cross Bio-Medical (Beijing) Co., Ltd.

55IC402-50

Span Diagnostics Ltd.

Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

W056-C

Guangzhou Wondfo Biotech Co. Ltd.

IR013025 G0111

J. Mitra & Co. Pvt. Ltd. Access Bio. Inc.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a Denotes no visible band b 4 (4%) of the 100 P. falciparum dilution samples sets had 200 and 5000 parasites/μL and 2 (6%) of the 35 P. vivax dilution sample sets had 200 and 5000 parasites/μL c Calculations include invalid tests

Annexes

99

10 0

Table A4.3: Distribution of pan/Pv test band intensity (0-4) scores for phase-2 wild-type P. vivax samples at low (200) and high (2000) parasite density (parasites/μL) 200 parasites/μL 200 parasites/μL Percentage distribution of Pv test band intensitye (n=140) 0a 1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 1.4 0.0 1.4 0.0 1.4 1.4 0.0 0.0 54.3 2.9 27.9 30.7 72.9 62.1 69.3 37.9 24.3 10.0 0.0 5.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.9 1.4 1.4 2.9 0.0 0.0 52.9 0.0 0.0 0.0 0.0 0.0 2.9 10.0 21.4 11.4 55.7 31.4 17.1 18.6 40.0 11.4 27.1 7.1 12.9 15.7 50.0 80.0 60.0 52.9 38.6 42.9 55.7 61.4 5.7 68.6 45.7 55.7 45.7 60.0 35.7 7.1 18.6 34.3 2.9 22.9 25.7 20.0 0.0 18.6 27.1 37.1 41.4 24.3 11.4 0.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

2000b parasites/μL Percentage distribution of pan test band intensitye (n=70) 0a 1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

2000b parasites/μL Percentage distribution of Pv test band intensitye (n=70) 0a 1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Product 0a 1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Catalogue number Manufacturer 2 3 4 2 3 4 2 3 4 Percentage distribution of pan test band intensitye (n=140)

2 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

3 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Pf only InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd NA NA NA NA NA NA NA NA NA

Advanced Quality™ One Step Malaria Pf Test

ITP11002TC1/ TC40 IR016025 G0141 KMFC6001

Avantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum

I13FRC

KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo 26.4 10.7 0.7 0.0 10.0 6.4 0.7 5.0 1.4 6.4 7.9 22.9 54.3 3.6 18.6 82.9 2.1 2.1 0.0 2.1 62.9 70.0 37.9 64.3 58.6 17.1 57.9 80.0 44.3 27.9 7.1 7.9 30.0 21.4 0.0 38.6 17.1 51.4 1.4 0.0 0.0 2.1 1.4 0.0 1.4 0.7 4.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 84.3 8.6 0.7 0.0 0.0 65.7 79.3 77.9 8.6 56.4 77.1 11.4 76.4 67.1 7.9 10.0 20.0 80.0 32.1 16.4 72.1 18.6 27.9 0.0 0.0 1.4 10.7 1.4 0.0 15.0 0.0 3.6 0.0 0.0 0.0 0.7 0.0 0.0 0.7 0.0 0.0 1.4 0.0 1.4 0.0 1.4 0.0 0.0 0.0 4.3 1.4 2.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 57.1 51.4 11.4 0.0 7.1 38.6 0.0 24.3 17.1 37.1 32.9 67.1 17.1 44.3 50.0 12.9 51.4 55.7 2.9 12.9 20.0 82.9 47.1 11.4 87.1 24.3 22.9 NA NA NA NA NA NA NA NA NA

0502_K25 KH-R-06-20 05FK50/05FK53 VB01

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131

Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd.

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria Pf/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test

NG-Test MALARIA Pf/Pan (pLDH)

OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MALW23-001 R0113C 50310025 IMPN-C52

Table A4.3 (continued) 200 parasites/μL 200 parasites/μL Percentage distribution of Pv test band intensitye (n=140) 0a 1 NA NA 64.3 82.1 76.4 27.9 57.9 60.7 NA NA 4.3 NA NA 10.7 70.7 1.4 0.0 0.0 NA NA 8.6 0.0 0.0 0.0 0.0 0.0 NA NA 10.0 0.0 0.0 8.6 0.0 0.0 0.0 2.9 1.4 0.0 7.1 1.4 NA NA 1.4 1.4 0.0 0.0 1.4 2.9 NA NA NA NA NA NA NA NA NA NA NA NA

2000b parasites/μL Percentage distribution of pan test band intensitye (n=70) 0a 1 0.0 30.0 NA NA NA NA NA NA 0.0 0.0 2.9 0.0 32.9 2.9 64.3 95.7 NA NA NA NA NA 35.0 NA NA NA 31.4 NA NA NA 0.0 NA NA NA 15.0 NA NA NA 7.9 NA NA NA 27.1 8.6 58.6 24.3 7.1 67.1 0.0 NA NA

2000b parasites/μL Percentage distribution of Pv test band intensitye (n=70) 0a 1 2 NA NA 40.0 50.0 45.7 2.9 35.7 55.7 NA NA

Product 0a 1 25.7 19.3 NA NA NA NA NA NA 15.0 0.7 72.9 67.1 10.7 25.0 1.4 4.3 0.0 1.4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 59.3 2.9 12.9 0.0 0.0 0.0 0.0 4.3

Catalogue number Manufacturer 2 3 4 2 3 4 2 3 4 Percentage distribution of pan test band intensitye (n=140) 3 2.1 77.9 NA NA NA NA NA NA 0.0 2.9

4 NA NA 55.7 41.4 41.4 55.7 51.4 32.9 NA NA NA NA 2.9 4.3 11.4 41.4 4.3 7.1 NA NA

05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc.

AM4650-K

SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device

GM006

MAT-PF/ PAN-50 523352

55IC402-50

Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a Denotes no visible band b 2 (6%) of the 35 P. vivax dilution sample sets had 200 and 5000 parasites/μL c Pan test line d P. vivax test line e Calculations include invalid tests

Annexes

101

10 2

Table A4.4: Panel detection score of phase-2 wild-type P. falciparum at low (200) and high (2000) parasite density (parasites/μL) by continent 200 parasites/μL Panel detection scorea by continent of sample origin Africa (n=58) InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 56.9 81.0 37.9 75.9 63.8 67.2 82.8 82.8 82.8 74.1 74.1 75.9 53.4 82.8 87.9 50.0 81.0 48.3 91.4 84.5 75.9 86.2 65.5 91.4 75.9 100.0 100.0 95.7 82.6 100.0 100.0 73.9 100.0 34.8 100.0 95.7 100.0 100.0 87.0 95.7 100.0 73.9 100.0 26.1 100.0 100.0 87.0 100.0 100.0 100.0 84.2 94.7 10.5 89.5 89.5 94.7 100.0 94.7 100.0 89.5 100.0 94.7 89.5 100.0 100.0 78.9 100.0 26.3 100.0 100.0 100.0 100.0 84.2 100.0 100.0 89.7 100.0 93.1 100.0 100.0 94.8 100.0 100.0 100.0 100.0 100.0 100.0 94.8 100.0 96.6 91.4 100.0 98.3 100.0 100.0 100.0 100.0 93.1 98.3 94.8 43.1 82.8 84.5 86.2 91.4 62.1 70.7 91.4 75.9 65.2 100.0 100.0 95.7 100.0 87.0 91.3 100.0 95.7 68.4 94.7 100.0 84.2 100.0 84.2 89.5 100.0 94.7 91.4 100.0 100.0 100.0 100.0 91.4 89.5 98.3 100 91.3 95.7 100.0 100.0 100.0 91.3 90.9 100 100 95.7 100.0 100.0 100.0 95.7 95.7 100.0 95.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.7 100.0 100.0 100.0 100.0 91.3 100.0 100.0

Product Asia (n=23) South America (n=19) Africa (n=58) Asia (n=23)

Catalogue number Manufacturer

2000b parasites/μL Panel detection scorea by continent of sample origin South America (n=19) 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 94.7 100.0 89.5 100.0 100.0 89.5 100.0 100.0 100.0 100.0 100.0 100.0 94.7 100.0 100.0 100.0 100.0 94.7 100.0 100.0 100.0 100.0 100.0 100.0 100.0

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

Table A4.4 (continued) 200 parasites/μL Panel detection scorea by continent of sample origin Africa (n=58) ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. 74.1 74.1 87.0 95.7 73.7 100.0 98.3 98.3 100.0 100.0 70.7 79.3 75.9 87.9 46.6 79.3 95.7 91.3 95.7 95.7 82.6 100.0 94.7 94.7 94.7 100.0 89.5 94.7 100.0 96.6 100.0 100.0 89.7 96.6 95.7 95.7 100.0 100.0 91.3 100.0

Product Asia (n=23) South America (n=19) Africa (n=58) Asia (n=23)

Catalogue number Manufacturer

2000b parasites/μL Panel detection scorea by continent of sample origin South America (n=19) 100.0 100.0 100.0 100.0 94.7 100.0 94.7 100.0

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Africa: Central African Republic, Ethiopia, Kenya, Madagascar, Nigeria, United Republic of Tanzania Asia: Cambodia, Myanmar, the Philippines South America: Colombia, Peru Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a A sample is considered detected only if all RDTs from both lots read by the first technician, at the minimum specified reading time, are positive b 4 (4%) of the 100 P. falciparum dilution samples sets had 200 and 5000 parasites/μL

Annexes

103

10 4

Table A4.5: Phase-2 P. falciparum test line false-positive rates for wild-type P. vivax samples at low (200) and high (2000) parasite density (parasites/µL) P. vivax samples (n=35) Manufacturer Lot 1 (n=70) InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 0.0 (69) 10.8 (65) 0.0 0.0 0.0 0.0 1.4 17.1 4.3 0.0 (69) 0.0 0.0 5.7 0.0 (69) 8.6 4.3 0.0 (68) 54.3 50.0 2.9 0.0 1.4 5.7 1.4 0.0 (69) 1.4 0.0 0.0 21.4 0.0 (69) 0.0 4.3 1.4 41.4 27.1 15.7 0.0 0.0 0.0 0.0 1.4 0.0 2.9 (69) 1.4 0.0 1.4 1.4 0.0 7.1 1.4 0.0 (139) 6.7 (134) 0.7 0.0 0.7 0.7 0.7 12.1 2.9 0.7 (139) 0.0 0.0 13.6 0.0 (138) 4.3 4.3 0.7 (138) 47.9 38.6 9.3 0.0 0.7 2.9 0.7 0.7 (139) 0.0 2.9 0.0 0.0 0.0 0.0 0.0 8.6 0.0 (34) 0.0 0.0 0.0 5.7 2.9 5.7 0.0 0.0 (34) 34.3 45.7 2.9 0.0 2.9 11.4 0.0 0.0 (34) 7.1 0.0 0.0 2.9 0.0 7.1 5.7 0.0 1.4 0.0 1.4 0.0 1.4 1.4 0.0 17.1 0.0 2.9 3.6 0.7 0.0 2.1 0.7 3.6 11.4 0.0 2.1 11.4 0.0 0.0 2.9 0.0 8.6 11.4 2.9 2.9

Product Lot 2 (n=70) Overall (n=140) Lot 1 (n=35)

Catalogue number Lot 2 (n=35) 0.0 0.0 0.0 0.0 0.0 0.0 14.3 2.9 0.0 0.0 0.0 0.0 0.0 (34) 0.0 0.0 0.0 5.7 3. (33) 0.0 0.0 0.0 8.6 0.0 0.0 0.0 0.0 37.1 34.3 5.7 2.9 0.0 0.0 2.9 0.0

200 parasites/μL False-positive Pf infectionb (%)

2000a parasites/μL False-positive Pf infectionb (%) Overall (n=70) 5.7 0.0 0.0 1.4 0.0 4.3 12.9 2.9 1.4 0.0 1.4 0.0 0.0 (69) 0.0 0.0 0.0 7.1 1.5 (67) 0.0 0.0 0.0 7.1 1.4 2.9 0.0 0.0 (69) 35.7 40.0 4.3 1.4 1.4 5.7 1.4 0.0 (69)

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

Pf only Advanced Quality™ One Step Malaria Pf Test Avantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

Table A4.5 (continued) P. vivax samples (n=35) Manufacturer Lot 1 (n=70) ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. Pvom, Plasmodium vivax, ovale and malariae NA NA NA NA NA NA NA NA NA NA 0.0 2.9 10.1 (69) 51.4 10.0 1.4 0.0 10.3 (68) 1.4 55.7 1.4 4.3 0.0 6.5 (138) 5.8 (139) 53.6 5.7 2.9 0.0 0.0 5.9 (34) 42.9 5.7 0.0 0.0 5.7 2.9 25.7 0.0 5.7

Product Lot 2 (n=70) Overall (n=140) Lot 1 (n=35) Lot 2 (n=35)

Catalogue number

200 parasites/μL False-positive Pf infectionb (%)

2000a parasites/μL False-positive Pf infectionb (%) Overall (n=70) 0.0 2.9 4.3 (69) 34.3 2.9 2.9 NA NA

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  a 2 (6%) of the 35 P. vivax dilution sample sets had 200 and 5000 parasites/μL b Pf line positive indicates a false-positive P. falciparum infection

Annexes

105

10 6

Table A4.6: Phase-2 pan (or P. vivax /\Pvom) test line false-positive rate for non-P. falciparum infection on phase-2 wild-type P. falciparum samples at low (200) and high (2000) parasite density (parasites/µL) P. falciparum samples (n=100) Manufacturer Lot 1 (n=200) InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 0.0 0.0 (190) 1.5 3.0 0.5 0.0 0.5 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 23.0 0.0 1.0 0.0 0.5 3.5 0.5 0.0 0.5 0.5 0.0 0.0 1.0 (197) 0.0 0.0 (199) 0.0 22.0 0.0 0.0 (199) 0.0 0.5 0.5 1.0 0.0 0.5 0.5 (199) 1.5 4.0 1.0 1.0 2.5 0.5 0.0 0.3 0.3 (389) 1.5 3.5 0.8 0.5 1.5 0.3 0.3 0.3 0.3 0.0 0.0 0.5 (396) 0.3 0.0 (398) 0.0 (398) 22.5 0.0 0.5 (399) 0.0 (399) 0.5 2.0 0.8 0.0 0.0 0.0 (97) 1.0 0.0 0.0 (98) 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 (99) 1.0 0.0 1.0 (99) NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Product Lot 2 (n=200) Overall (n=400) Lot 1 (n=100)

Catalogue number

200 parasites/μL False-positive non-Pf infection (%)

2000a parasites/μL False-positive non-Pf infection (%) Lot 2 (n=100) NA NA NA NA NA NA NA NA NA 0.0 (99) 0.0 0.0 0.0 1.0 1.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 (99) 0.0 0.0 (99) 0.0 (99) 2.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0

Overall (n=200) NA NA NA NA NA NA NA NA NA 0.0 (199) 0.0 (197) 0.5 0.0 0.5 (198) 2.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 (199) 0.5 0.0 (199) 0.0 (199) 1.5 0.0 0.0 0.0 0.0 (199) 0.5 0.5 0.5 (199)

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

Table A4.6 (continued) P. falciparum samples (n=100) Manufacturer Lot 1 (n=200) ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. NA NA NA NA NA NA NA NA NA NA 18.5 1.5 23.6 20.5 3.5 0.5 14.5 1.5 (197) 31.0 22.5 0.5 2.5 (199) 16.5 1.5 (391) 27.3 (399) 21.5 2.0 (399) 1.5 (399) 91.0 3.1 (96) 86.9 (99) 10.0 1.0 0.0 80.0 4.0 (99) 88.0 8.0 0.0 3.0

Product Lot 2 (n=200) Overall (n=400) Lot 1 (n=100) Lot 2 (n=100)

Catalogue number

200 parasites/μL False-positive non-Pf infection (%)

2000a parasites/μL False-positive non-Pf infection (%) Overall (n=200) 85.5 3.6 (195) 87.4 (199) 9.0 0.5 1.5 NA NA

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a 4 (4%) of the 100 P. falciparum dilution samples sets had 200 and 5000 parasites/μL

Annexes

107

10 8

Table A4.7: Phase-2 false-positive rate for P. falciparum test line results on all malaria-negative samples Percentage of false-positive Pf test lines on “cleana” negative samples Lot 1 (n=118) InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 12.0 (117) 11.5 (113) 0.0 0.0 0.0 0.8 0.0 7.6 2.5 0.8 0.0 0.0 (117) 3.4 0.9 (117) 3.4 1.7 0.9 (116) 37.6 (117) 39.8 5.1 0.8 3.4 10.2 0.0 0.9 (117) 0.0 0.0 15.3 0.8 0.8 0.0 0.0 (116) 35.6 37.3 20.3 1.7 4.2 0.8 0.0 0.8 1.7 (117) 1.3 (235) 0.0 0.0 (235) 9.3 0.9 (235) 2.1 0.8 0.4 (232) 36.6 (235) 38.6 12.7 1.3 3.8 5.5 0.0 0.9 (235) 0.0 0.0 0.0 12.0 (25) 0.0 7.7 0.0 0.0 (25) 73.1 42.3 3.8 0.0 0.0 0.0 0.0 0.0 2.6 (117) 5.9 0.9 0.0 0.0 0.0 0.8 15.3 0.8 7.3 (234) 8.7 (231) 0.4 0.0 0.0 0.4 0.4 11.4 1.7 23.1 0.0 0.0 0.0 0.0 0.0 3.8 7.7 3.8 11.5 0.0 0.0 0.0 (25) 0.0 0.0 0.0 26.9 0.0 11.5 0.0 0.0 34.6 0.0 (25) 0.0 0.0 0.0 65.4 11.5 23.1 0.0 3.8 0.0 0.0 0.0 17.3 0.0 0.0 0.0 (51) 0.0 0.0 1.9 17.3 1.9 5.8 0.0 0.0 23.5 (51) 0.0 (51) 3.8 0.0 0.0 (51) 69.2 26.9 13.5 0.0 1.9 0.0 0.0 0.0 13.9 (115) 0.0 0.0 0.8 0.0 10.3 11.1 (117) 0.0 10.2 1.7 0.0 1.7 0.9 (117) 0.8 0.0 10.2 0.0 0.0 7.7 (233) 0.0 0.8 0.9 (235) 0.4 5.1 10.6 (235) 0.0 5.1 0.0 0.0 0.0 3.8 0.0 0.0 0.0 0.0 19.2 0.0 0.0 0.0 0.0 0.0 0.0 23.1 0.0 7.7 0.0 0.0 0.0 1.9 0.0 0.0 11.5 0.0 13.5 3.5 6.9 3.4 0.0 0.0 13.8 8.6 0.0 10.3 12.1 0.0 0.0 0.0 5.2 0.0 5.2 34.5 12.1 3.6 (56) 0.0 7.0 (57) 6.9 10.3 12.1 29.3 3.4 81.0 48.3 8.6 0.0 0.0 3.4 3.4 3.4

Product Lot 2 (n=118) Overall (n=236) Lot 1 (n=26) Lot 2 (n=26) Overall (n=52) Lot 1 (n=58)

Catalogue number Manufacturer

Percentage of false-positive Pf test lines on samples containing non-Plasmodium spp. infectious agentsb Lot 2 (n=58) 1.7 5.2 3.4 0.0 0.0 13.8 17.2 1.7 0.0

Percentage of false-positive Pf test lines on samples containing immunological factorsc Overall (n=116) 2.6 6.0 3.4 0.0 0.0 13.8 12.9 0.9 5.2 12.3 (57) 1.7 0.0 0.0 5.2 3.4 1.7 25.9 8.6 3.4 0.0 6.9 19.0 10.3 10.3 20.7 5.3 (57) 79.3 53.4 36.2 5.2 0.0 0.0 3.4 3.4 12.2 (115) 0.9 0.0 0.0 5.2 1.7 3.4 30.2 10.3 3.5 (114) 0.0 7.0 (115) 12.9 10.3 11.2 25.0 4.3 (115) 80.2 50.9 22.4 2.6 0.0 1.7 3.4 3.4

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

Table A4.7 (continued) Percentage of false-positive Pf test lines on “cleana” negative samples Lot 1 (n=118) ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. 0.9 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.5 1.3 0.0 0.0 0.0 9.3 0.0 4.7 0.0 0.0 0.0 29.3 0.0 0.0 6.9 0.0 (117) 0.9 (115) 3.5 (114) 73.7 0.8 0.0 (117) 1.7 76.3 0.4 (235) 0.4 (232) 2.6 (232) 75.0 0.0 (25) 0.0 (23) 0.0 (25) 46.2 0.0 0.0 (23) 7.7 42.3 0.0 (51) 0.0 (46) 3.9 (51) 44.2 5.2 5.4 (56) 20.7 56.9 6.9 5.4 (56) 19.0 67.2 27.6 0.0 0.0 8.6

Product Lot 2 (n=118) Overall (n=236) Lot 1 (n=26) Lot 2 (n=26) Overall (n=52) Lot 1 (n=58) Lot 2 (n=58)

Catalogue number Manufacturer

Percentage of false-positive Pf test lines on samples containing non-Plasmodium spp. infectious agentsb

Percentage of false-positive Pf test lines on samples containing immunological factorsc Overall (n=116) 6.0 5.4 (112) 19.8 62.1 28.4 0.0 0.0 7.8

AM4650-K GM006 MAT-PF/PAN-50 523352

55IC402-50

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium spp.  Pvom, Plasmodium vivax, ovale and malariae a Blood samples from healthy volunteers with no known current illness or blood abnormality b See Table A4.8 for details c See Table A4.9 for details

Annexes

109

11 0

Table A4.8: Phase-2 false-positive rate for P. falciparum in samples containing specific non-malarial infectious pathogens Percentage of false-positives for Plasmodium spp. by infectious pathogen Catalogue number Manufacturer Dengue Lot 1 (n=12) ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01 MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE RK MAL 001 I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499 0.0 0.0 0.0 9.1 (11) 0.0 0.0 0.0 0.0 100.0 58.3 8.3 0.0 0.0 8.3 0.0 0.0 8.3 0.0 0.0 41.7 0.0 0.0 0.0 0.0 100.0 25.0 33.3 8.3 8.3 25.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 8.3 0.0 8.3 0.0 0.0 0.0 0.0 0.0 16.7 0.0 8.3 0.0 60.0 0.0 0.0 0.0 0.0 0.0 0.0 20.0 0.0 0.0 0.0 0.0 0.0 0.0 20.0 0.0 0.0 (9) 100.0 30.0 0.0 0.0 10.0 0.0 0.0 0.0 30.0 0.0 0.0 0.0 (9) 0.0 0.0 0.0 40.0 0.0 10.0 0.0 0.0 40.0 0.0 (9) 0.0 0.0 0.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 0.0 0.0 0.0 8.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 25.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 50.0 0.0 0.0 0.0 0.0 0.0 0.0 20.0 0.0 20.0

Product Leishmaniasis Lot 1 (n=10) Lot 2 (n=10) Lot 2 (n=12)

Chagas Lot 1 (n=4) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 50.0 0.0 0.0 0.0 0.0 100.0 25.0 0.0 0.0 0.0 0.0 0.0 0.0

Lot 2 (n=4) 0.0 0.0 0.0 0.0 0.0 0.0 25.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 50.0 0.0 25.0 0.0 0.0 50.0 0.0 0.0 0.0 0.0 100.0 0.0 50.0 0.0 0.0 0.0 0.0 0.0

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom

Table A4.8 (continued) Percentage of false-positives for Plasmodium spp. by infectious pathogen Catalogue number Manufacturer Dengue Lot 1 (n=12) AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C IR013025 G0111 Pvom, Plasmodium vivax, ovale and malariae J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. 0.0 0.0 8.3 75.0 0.0 0.0 0.0 0.0 (10) 8.3 58.3 0.0 0.0 0.0 (9) 0.0 (7) 0.0 (9) 20.0 0.0 0.0 0.0 0.0 (9) 10.0 10.0 0.0 0.0 0.0 0.0 0.0 50.0 0.0 0.0

Product Leishmaniasis Lot 1 (n=10) Lot 2 (n=10) Lot 1 (n=4) Chagas Lot 2 (n=12)

Lot 2 (n=4) 0.0 0.0 25.0 100.0 0.0 0.0 0.0 0.0

ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Pf, Plasmodium falciparum 

Pv, Plasmodium vivax 

pan, Plasmodium spp. 

Annexes

111

11 2

Table A4.9: Phase-2 false-positives rate for P. falciparum in samples containing potentially cross-reacting blood immunological factors Percentage of false-positive for Plasmodium spp. by blood immunological factor Manufacturer Rheumatoid factor Lot 1 (n=12) Lot 2 (n=12) Lot 1 (n=26) Lot 2 (n=26) InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 58.3 0.0 0.0 0.0 25.0 66.7 16.7 75.0 16.7 8.3 0.0 66.7 66.7 8.3 83.3 75.0 16.7 100.0 83.3 91.7 41.7 0.0 50.0 0.0 58.3 8.3 0.0 58.3 75.0 8.3 83.3 66.7 16.7 100.0 83.3 83.3 33.3 0.0 50.0 0.0 50.0 0.0 (25) 0.0 0.0 (25) 7.7 7.7 11.5 19.2 0.0 100.0 50.0 15.4 0.0 0.0 15.4 0.0 7.7 58.3 0.0 0.0 0.0 16.7 50.0 33.3 83.3 16.7 3.9 0.0 0.0 0.0 0.0 0.0 0.0 19.2 11.5 3.9 3.9 0.0 0.0 3.9 3.9 0.0 7.7 11.5 7.7 0.0 3.9 30.8 7.7 15.4 7.7 0.0 (25) 96.2 57.7 42.3 19.2 0.0 11.5 0.0 7.7 66.7 33.3 0.0 0.0 33.3 16.7 33.3 100.0 66.7 0.0 0.0 33.3 33.3 66.7 33.3 33.3 66.7 100.0 83.3 66.7 0.0 0.0 16.7 33.3 33.3 0.0 16.7 0.0 0.0 0.0 25.0 0.0 0.0 25.0 0.0 8.3 0.0 0.0 0.0 33.3 16.7 0.0 0.0 7.7 0.0 0.0 0.0 0.0 3.9 7.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 11.5 0.0 0.0 0.0 33.3 33.3 0.0 0.0 66.7 50.0 0.0 33.3 16.7 33.3 33.3 0.0 0.0 66.7 50.0 16.7 0.0 40.0 (5) 16.7 0.0 0.0 50.0 0.0 33.3 100.0 66.7 16.7 0.0 33.3 33.3 66.7 66.7 33.3 66.7 66.7 66.7 66.7 16.7 0.0 16.7 33.3 33.3

Product Anti-nuclear antibodies Lot 1 (n=6) Lot 2 (n=6) Anti-mouse antibodies

Catalogue number

Rapid plasma reagin Lot 1 (n=14) Lot 2 (n=14) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.1 0.0 0.0 0.0 0.0 0.0 0.0 14.3 14.3 0.0 7.7 (13) 0.0 0.0 0.0 0.0 14.3 7.1 0.0 100.0 35.7 0.0 0.0 0.0 14.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 14.3 0.0 0.0 14.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 100.0 50.0 28.6 7.1 0.0 7.1 0.0 0.0

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums & Vaccines Ltd. ) Premier Medical Corporation GenBody Inc. Green Cross Medical Science Corp. (Korea) Humasis Co., Ltd. ICT INTERNATIONAL Artron Laboratories Inc. Zhejiang Orient Gene Biotech Co., Ltd. AZOG, INC. Medical Diagnostech (Pty) Ltd SARL NG Biotech, Z.A. CTK Biotech Inc. Zephyr Biomedicals Hangzhou Biotest Biotech Co. Ltd. Standard Diagnostics Inc. IMACCESS S.A.S

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

RK MAL 001

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

I16FRC MALAG100 20-0146-01 AMAL-7025 ML03 A03-18-322 RT 20222 MFV-124 MDMALLDH001 NG-MAL-W23-001 R0113C 50310025 IMPN-C52 05FK60/05FK63 412499

Table A4.9 (continued) Percentage of false-positive for Plasmodium spp. by blood immunological factor Manufacturer Rheumatoid factor Lot 1 (n=12) Lot 2 (n=12) Lot 1 (n=26) Lot 2 (n=26) ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. Pvom, Plasmodium vivax, ovale and malariae 0.0 16.7 0.0 25.0 0.0 0.0 0.0 0.0 0.0 33.3 0.0 33.3 66.7 66.7 58.3 75.0 75.0 0.0 58.3 58.3 66.7 83.3 83.3 0.0 0.0 8.0 (25) 0.0 69.2 11.5 0.0 0.0 12.5 (24) 7.7 84.6 7.7 0.0 33.3 33.3 100.0 66.7 100.0 0.0 50.0 16.7 66.7 33.3 66.7 0.0 0.0 15.4 (13) 0.0 64.3 0.0 0.0 0.0 0.0

Product Anti-nuclear antibodies Lot 1 (n=6) Lot 2 (n=6) Anti-mouse antibodies

Catalogue number

Rapid plasma reagin Lot 1 (n=14) Lot 2 (n=14) 0.0 7.1 0.0 64.3 0.0 0.0 0.0 0.0

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Pf, Plasmodium falciparum 

Pv, Plasmodium vivax 

pan, Plasmodium species 

Annexes

113

11 4

Table A4.10: Phase-2 false-positive rate of pan, P. vivax or Pvom test line results in all malaria-negative samples Percentage of false-positive pan test lines on “cleana” negative samples Percentage of false-positive pan test lines on samples containing non-Plasmodium spp. infectious agentsb Lot 1 (n=26) NA NA NA NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (235) 0.0 0.0 (235) 4.2 0.4 (235) 1.7 0.9 0.0 (232) 77.9 (235) 4.2 4.7 0.0 0.4 10.6 0.0 0.9 (235) 0.0 0.0 0.0 4.0 (25) 0.0 0.0 0.0 0.0 (25) 100.0 0.0 0.0 0.0 3.9 3.9 0.0 0.0 3.9 0.0 0.0 0.0 (25) 0.0 7.7 0.0 0.0 0.0 3.9 0.0 0.0 26.9 0.0 (25) 0.0 0.0 0.0 100.0 0.0 0.0 3.9 0.0 11.5 0.0 0.0 1.9 0.0 0.0 0.0 (51) 0.0 3.9 0.0 0.0 0.0 1.9 0.0 0.0 15.7 (51) 0.0 (51) 0.0 0.0 0.0 (51) 100.0 0.0 0.0 1.9 1.9 7.7 0.0 0.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 19.0 3.5 0.0 0.0 8.6 15.5 6.9 36.2 6.9 3.6 (56) 0.0 15.8 (57) 17.2 6.9 25.9 22.4 10.3 100.0 27.6 32.8 8.6 0.0 20.7 0.0 19.0

Product Lot 1 (n=118)Lot 2 (n=118) InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 10.3 (117) 1.8 (113) 0.0 0.0 6.8 0.0 0.0 4.2 0.9 0.0 0.0 0.0 (117) 2.5 0.9 (117) 2.5 1.7 0.0 (116) 87.2 (117) 3.4 4.2 0.0 0.0 11.9 0.0 1.7 (117) 0.0 0.0 5.9 0.0 0.9 0.0 0.0 (116) 68.6 5.1 5.1 0.0 0.9 9.3 0.0 0.0 0.0 (117) 0.0 (117) 0.0 0.0 0.0 2.5 0.0 0.0 15.3 0.9 5.1 (234) 0.9 (231) 0.0 0.0 4.7 0.0 0.0 9.8 0.9 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Catalogue number Manufacturer Overall (n=236) Lot 2 (n=26) Overall (n=52) Lot 1 (n=58)

Percentage of false-positive pan test lines on samples containing immunological factorsc Lot 2 (n=58) NA NA NA NA NA NA NA NA NA 17.5 (57) 1.7 0.0 0.0 10.3 8.6 8.6 31.0 10.3 8.6 0.0 15.5 27.6 5.2 29.3 20.7 10.5 (57) 93.1 31.0 29.3 13.8 0.0 19.0 0.0 17.2

Overall (n=116) NA NA NA NA NA NA NA NA NA 18.3 (115) 2.6 0.0 0.0 9.5 12.1 7.8 33.6 8.6 6.1 (114) 0.0 15.7 (115) 22.4 6.0 27.6 21.6 10.4 (115) 96.6 29.3 31.0 11.2 0.0 19.8 0.0 18.1

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

Pf only Advanced Quality™ One Step Malaria Pf Test Avantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of RK MAL 001 Bharat Serums & Vaccines Ltd. ) I16FRC Premier Medical Corporation MALAG100 GenBody Inc. 20-0146-01 Green Cross Medical Science Corp. (Korea) AMAL-7025 Humasis Co., Ltd. ML03 ICT INTERNATIONAL A03-18-322 Artron Laboratories Inc. RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd NG-MAL-W23-001 SARL NG Biotech, Z.A. R0113C CTK Biotech Inc. 50310025 Zephyr Biomedicals IMPN-C52 Hangzhou Biotest Biotech Co. Ltd. 05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S

Table A4.10 (continued) Percentage of false-positive pan test lines on “cleana” negative samples Percentage of false-positive pan test lines on samples containing non-Plasmodium spp. infectious agentsb Lot 1 (n=26) 0.0 (25) 0.0 (23) 4.0 (25) 50.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.9 0.0 (23) 3.9 42.3 0.0 0.0 0.0 (46) 3.9 (51) 46.2 0.0 0.0 23.2 (56) 17.2 65.5 6.9 0.0 0.0 0.0 (51) 17.2 15.5 19.6 (56) 20.7 69.0 1.7 0.0 0.0 8.6

Product Lot 1 (n=118)Lot 2 (n=118) ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. 0.9 0.0 0.0 0.0 0.4 0.0 0.9 (115) 0.0 (114) 66.1 3.4 0.0 0.0 (117) 0.9 68.6 0.0 2.5 0.4 (232) 0.4 (232) 67.4 1.7 1.3 0.0 (117) 0.0 0.0 (235)

Catalogue number Manufacturer Overall (n=236) Lot 2 (n=26) Overall (n=52) Lot 1 (n=58) Lot 2 (n=58)

Percentage of false-positive pan test lines on samples containing immunological factorsc Overall (n=116)

AM4650-K

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

16.4 21.4 (112) 19.0 67.2 4.3 0.0 0.0 7.7

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

NA, not applicable Pf, Plasmodium falciparum  Pv, Plasmodium vivax  pan, Plasmodium species  Pvom, Plasmodium vivax, ovale and malariae a Blood samples from healthy volunteers with no known current illness or blood abnormality b See Table A4.8 for details c See Table A4.9 for details

Annexes

115

No. invalid

No. invalid

No. invalid

No. invalid

No. invalid

No. positive

No. positive

No. positive

No. positive

No. positive

Mean band intensity

Mean band intensity

Mean band intensity

Mean band intensity

Mean band intensity

No. positive

No. invalid 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 2.0 2.7 2.3 3.0 1.0 1.7 2.1 2.0 13.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 ND 1.2 ND 1.9 2.1 1.8 2.7 2.0 1.0 3.0 15.0 0.0 15.0 14.0 13.0 14.0 15.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.5 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 2.0 1.7 2.0 ND 2.0 1.0 2.0 2.0 2.0 2.0 1.9 15.0 14.0 15.0 15.0 15.0 0.0 15.0 10.0 15.0 15.0 15.0 15.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01 10.0 14.0 0.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 0.0 15.0 6.0 15.0 15.0 15.0 15.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.9 1.4 2.0 1.9 2.0 ND 1.9 1.0 2.0 2.0 2.0 2.1 1.2 0.0 1.9 15.0 15.0 15.0 15.0 15.0 15.0 3.0 15.0 8.0 15.0 15.0 15.0 15.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 1.9 1.3 2.0 2.0 2.0 1.0 1.9 1.0 2.0 2.0 1.9 2.9 1.1 15.0 15.0 15.0 15.0 15.0 15.0 2.0 15.0 11.0 15.0 15.0 15.0 15.0 15.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 1.4 ND 2.0 2.9 2.0 2.1 2.7 1.7 15.0 12.0 0.0 15.0 15.0 15.0 15.0 15.0 15.0 0.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.8 ND 2.0 2.1 2.0 2.0 2.0 2.0 10.0 14.0 0.0 15.0 15.0 14.0 15.0 15.0 15.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 1.8 ND 2.0 2.0 1.9 2.0 2.1 1.1 1.8 1.9 1.0 2.1 2.0 2.1 1.0 1.9 1.0 2.0 2.0 1.7 3.0 1.0 12.0 15.0 0.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 0.0 15.0 6.0 15.0 15.0 15.0 15.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.9 ND 1.9 2.0 2.0 2.1 2.0 1.9 2.0 1.8 1.3 2.0 2.3 2.0 ND 2.1 1.0 2.0 2.1 1.8 2.9 2.0

InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd

14.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0

1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.1 2.6 2.3 2.6 1.9 2.0 2.2 1.7

14.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0

1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.0 2.0 2.3 3.0 1.8 2.0 2.2 1.9

14.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.0 2.7 3.0 2.5 1.0 1.9 2.3 1.7

15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.0 2.0 2.7 2.5 3.0 1.0 2.0 2.1 1.9

14.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 15.0 0.0 15.0 0.0 15.0 15.0 14.0 15.0 15.0 14.0 15.0 15.0 14.0 15.0 15.0 15.0 0.0 14.0 10.0 15.0 15.0 15.0 15.0 15.0

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat RK MAL 001 Serums & Vaccines Ltd. ) I16FRC Premier Medical Corporation MALAG100 GenBody Inc. 20-0146-01 Green Cross Medical Science Corp. (Korea) AMAL-7025 Humasis Co., Ltd. ML03 ICT INTERNATIONAL A03-18-322 Artron Laboratories Inc. RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd NG-MAL-W23-001 SARL NG Biotech, Z.A. R0113C CTK Biotech Inc. 50310025 Zephyr Biomedicals IMPN-C52 Hangzhou Biotest Biotech Co. Ltd.

Mean band intensity 1.0 2.0 2.2 3.0 2.6 1.0 2.0 2.4 1.7 1.0 1.2 ND 1.8 1.7 1.2 1.9 2.0 1.0 1.4 1.9 1.0 1.9 1.0 1.9 ND 2.0 1.0 2.0 2.0 1.6 2.0 1.9

11 6

Table A4.11: Heat stability testing results for P. falciparum (or pana) test line on a P. falciparum sample at low parasite density (200 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Baseline testing Lot 1 (n=15) Lot 2 (n=15) Lot 1 (n=15) Lot 2 (n=15) Lot 1 (n=15) Manufacturer 35 °C 45 °C Lot 2 (n=15)

Product

Catalogue number

Table A4.11 (continued) Baseline testing Lot 1 (n=15) No. invalid No. invalid No. invalid No. invalid No. invalid No. positive No. positive No. positive No. positive No. positive Mean band intensity Mean band intensity Mean band intensity Mean band intensity Mean band intensity No. positive

35 °C Lot 1 (n=15) Lot 2 (n=15) Lot 1 (n=15)

45 °C Lot 2 (n=15) No. invalid 15.0 14.0 1.0 1.8 2.0 2.9 1.0 2.0 1.0 1.0 8.0 15.0 0.0 0.0 1.0 1.0 11.0 13.0 15.0 15.0 15.0 13.0 10.0 15.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 Mean band intensity 2.3 1.0 1.0 1.8 2.0 2.0 1.9 1.5 1.0 1.0

Lot 2 (n=15)

Product

Catalogue number Manufacturer

05FK60/05FK63 412499 ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. Pvom, Plasmodium vivax, ovale and malariae 5.0 15.0 0.0 0.0 1.0 1.0 6.0 15.0 0.0 0.0 1.0 1.0 11.0 15.0 0.0 0.0 1.0 1.0 9.0 15.0 0.0 0.0 15.0 10.0 15.0 15.0 15.0 15.0 0.0 1.0 0.0 0.0 0.0 0.0 1.1 2.0 2.0 2.2 1.9 1.9 15.0 15.0 15.0 15.0 15.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 2.0 1.9 2.3 1.0 1.9 14.0 12.0 15.0 15.0 14.0 15.0 0.0 1.0 0.0 0.0 0.0 0.0 1.0 2.0 1.7 2.1 1.1 1.9 15.0 15.0 15.0 15.0 15.0 14.0 0.0 0.0 0.0 0.0 0.0 0.0 1.1 1.9 1.9 2.2 1.1 2.0 8.0 12.0 15.0 15.0 14.0 15.0 0.0 1.0 0.0 0.0 0.0 0.0

Standard Diagnostics Inc. IMACCESS S.A.S

15.0 15.0

0.0 0.0

1.9 1.1

15.0 15.0

0.0 0.0

2.2 1.8

15.0 15.0

0.0 0.0

2.1 1.0

15.0 14.0

0.0 1.0

2.0 1.0

15.0 15.0

0.0 0.0

2.0 1.0

SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

ND, not determined Pf, Plasmodium falciparum  a For pan-only tests

Pv, Plasmodium vivax 

pan, Plasmodium species 

Annexes

117

No. invalid

No. invalid

No. invalid

No. invalid

No. invalid

No. positive

No. positive

No. positive

No. positive

No. positive

Mean band intensity

Mean band intensity

Mean band intensity

Mean band intensity

Mean band intensity

No. positive

No. invalid 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 ND ND ND ND ND 1.0 1.0 1.0 ND 0.0 0.0 0.0 8.0 0.0 12.0 7.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ND ND ND ND ND ND ND 1.0 ND ND ND 1.0 1.0 ND ND 0.0 0.0 4.0 0.0 0.0 0.0 0.0 15.0 0.0 0.0 1.0 0.0 15.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette 1.0 0.0 0.0 10.0 0.0 0.0 13.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 0.0 2.0 0.0 0.0 0.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ND ND 2.0 ND ND ND ND 1.0 ND ND ND 1.0 ND ND ND 0.0 0.0 0.0 0.0 0.0 3.0 0.0 1.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ND ND ND ND ND 1.0 ND 1.0 ND ND ND 1.0 ND ND ND 3.0 0.0 0.0 0.0 0.0 1.0 0.0 14.0 0.0 2.0 0.0 1.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA 0.0 ND 0.0 0.0 ND 0.0 0.0 ND 1.0 ND ND ND ND 1.0 ND 1.0 ND 1.0 ND 1.0 1.0 ND ND NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 14.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 NA NA NA NA NA NA ND ND ND ND ND ND ND ND 1.0 ND ND ND ND 1.0 ND ND NA NA NA NA NA NA 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 ND ND 1.0 ND ND 1.0 ND ND 0.0 0.0 0.0 14.0 0.0 0.0 15.0 0.0 0.0 0.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ND ND ND 1.0 ND ND 1.0 ND ND 3.0 0.0 0.0 15.0 1.0 0.0 15.0 2.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 ND ND 1.0 1.0 ND 1.0 1.0 ND 0.0 0.0 0.0 13.0 15.0 0.0 11.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ND ND ND 1.0 1.0 ND 1.0 1.0 ND

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

0.0 0.0 0.0 0.0 0.0 8.0 9.0 1.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 15.0 0.0 0.0 0.0 4.0 15.0 0.0 0.0 NA NA NA NA NA NA

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd.

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat RK MAL 001 Serums & Vaccines Ltd. ) I16FRC Premier Medical Corporation MALAG100 GenBody Inc. 20-0146-01 Green Cross Medical Science Corp. (Korea) AMAL-7025 Humasis Co., Ltd. ML03 ICT INTERNATIONAL A03-18-322 Artron Laboratories Inc. RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd NG-MAL-W23-001 SARL NG Biotech, Z.A. R0113C CTK Biotech Inc. 50310025 Zephyr Biomedicals IMPN-C52 Hangzhou Biotest Biotech Co. Ltd. 05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

ND, not determined NA, not applicable Pf, Plasmodium falciparum 

Pv, Plasmodium vivax 

pan, Plasmodium species 

Pvom, Plasmodium vivax, ovale and malariae

Mean band intensity ND ND ND 1.0 ND 1.0 1.0 ND ND ND ND ND 1.3 ND ND ND ND 1.0 ND ND 1.0 ND 1.0 ND ND NA NA NA NA NA NA

11 8

Table A4.11a: Heat stability testing results for pan test line of combination RDTs on a P. falciparum sample at low parasite density (20 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Baseline testing Lot 1 (n=15) Lot 2 (n=15) Lot 1 (n=15) Lot 2 (n=15) Lot 1 (n=15) Manufacturer 35 °C 45 °C Lot 2 (n=15)

Product

Catalogue number

Table A4.12: Heat stability testing results for P. falciparum (or pana) test line on a P. falciparum sample at high parasite density (2000 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Baseline testing Lot 1 (n=5) No. invalid No. invalid No. invalid No. invalid No. invalid No. positive No. positive No. positive No. positive No. positive Mean band intensity Mean band intensity Mean band intensity Mean band intensity Mean band intensity

35 °C Lot 1 (n=5) Lot 2 (n=5) Lot 1 (n=5)

45 °C Lot 2 (n=5) No. positive No. invalid 2.2 4.0 4.0 4.0 4.0 3.0 4.0 4.0 4.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.4 3.6 1.0 3.8 4.0 4.0 4.0 4.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 5.0 0.0 4.0 3.2 3.0 3.0 4.0 1.2 4.0 1.0 4.0 4.0 4.0 4.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Mean band intensity 2.0 4.0 4.0 4.0 4.0 3.0 4.0 4.0 4.0 1.8 3.0 1.0 4.0 3.4 3.8 4.0 4.0 3.0 3.2 4.0 2.8 2.4 3.0 4.0 1.0 3.8 1.0 3.0 4.0 4.0 4.0 3.0

Lot 2 (n=5)

Product

Catalogue number Manufacturer

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01 5.0 5.0 5.0 5.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 3.4 4.0 4.0 4.0 1.6 4.0 1.0 4.0 4.0 3.4 4.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 4.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.8 4.0 3.0 4.0 4.0 4.0 1.8 4.0 1.0 4.0 4.0 4.0 4.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 2.6 3.0 1.0 4.0 4.0 4.0 4.0 4.0 3.8 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 3.0 1.0 4.0 4.0 4.0 4.0 4.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 3.6 1.0 4.0 4.0 3.8 4.0 4.0 4.0 4.0 4.0 3.0 4.0 4.0 4.0 1.8 4.0 1.0 4.0 4.0 3.8 4.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 2.6 3.0 1.0 4.0 4.0 3.8 4.0 4.0 4.0 4.0 4.0 3.0 3.4 4.0 4.0 2.0 4.0 1.0 4.0 4.0 3.8 4.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

2.2 4.0 4.0 4.0 4.0 3.0 4.0 3.8 4.0

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

3.0 4.0 4.0 4.0 4.0 3.0 4.0 4.0 4.0

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

2.6 4.0 4.0 4.0 4.0 3.2 4.0 4.0 4.0

3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

2.0 3.8 4.0 4.0 4.0 2.2 4.0 4.0 4.0

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat RK MAL 001 Serums & Vaccines Ltd. ) I16FRC Premier Medical Corporation MALAG100 GenBody Inc. 20-0146-01 Green Cross Medical Science Corp. (Korea) AMAL-7025 Humasis Co., Ltd. ML03 ICT INTERNATIONAL A03-18-322 Artron Laboratories Inc. RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd NG-MAL-W23-001 SARL NG Biotech, Z.A. R0113C CTK Biotech Inc. 50310025 Zephyr Biomedicals IMPN-C52 Hangzhou Biotest Biotech Co. Ltd.

(continued)

Annexes

119

No. invalid

No. invalid

No. invalid

No. invalid

No. invalid

No. positive

No. positive

No. positive

No. positive

No. positive

Mean band intensity

Mean band intensity

Mean band intensity

Mean band intensity

Mean band intensity

No. positive

No. invalid 0.0 0.0 4.0 2.6 5.0 5.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 2.2 3.0 3.0 4.0 4.0 4.0 5.0 4.0 5.0 5.0 5.0 5.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 2.3 2.0 5.0 5.0 0.0 0.0

05FK60/05FK63 412499 ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. Pvom, Plasmodium vivax, ovale and malariae 5.0 5.0 0.0 0.0 1.8 2.4 5.0 5.0 0.0 0.0 2.0 2.0 5.0 5.0 0.0 0.0 2.0 2.0 5.0 5.0 0.0 0.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 4.0 3.2 4.0 3.2 3.8 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 4.0 3.4 4.0 3.8 4.0 5.0 4.0 5.0 5.0 5.0 5.0 0.0 1.0 0.0 0.0 0.0 0.0 2.8 3.3 4.0 3.6 3.6 4.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 3.6 3.0 4.0 4.0 3.4 2.0 2.0 5.0 3.0 5.0 5.0 4.0 5.0 4.0 5.0

Standard Diagnostics Inc. IMACCESS S.A.S

5.0 5.0

0.0 0.0

4.0 2.8

5.0 5.0

0.0 0.0

4.0 3.8

5.0 5.0

0.0 0.0

4.0 3.0

5.0 5.0

0.0 0.0

3.8 2.2

5.0 5.0

SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

ND, not determined Pf, Plasmodium falciparum  a For pan-only tests

Pv, Plasmodium vivax 

pan, Plasmodium species 

Mean band intensity 4.0 2.6 2.0 3.0 3.0 4.0 4.0 4.0 1.8 2.0

12 0

Table A4.12: Heat stability testing results for P. falciparum (or pana) test line on a P. falciparum sample at high parasite density (2000 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C (continued) Baseline testing 45 °C Lot 2 (n=5) Lot 1 (n=5) Lot 2 (n=5) Lot 1 (n=5) Manufacturer Lot 2 (n=5) Lot 1 (n=5) 35 °C

Product

Catalogue number

Table A4.12a: Heat stability testing results for pan test line of combination RDTs on a P. falciparum sample at high parasite density (2000 parasites/μL). Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Baseline testing Lot 1 (n=5) No. invalid No. invalid No. invalid No. invalid No. invalid No. positive No. positive No. positive No. positive No. positive Mean band intensity Mean band intensity Mean band intensity Mean band intensity Mean band intensity

35 °C Lot 1 (n=5) Lot 2 (n=5) Lot 1 (n=5)

45 °C Lot 2 (n=5) No. positive No. invalid ND 1.0 1.0 1.6 1.0 1.0 1.8 1.0 1.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 5.0 0.0 1.0 1.0 ND 1.0 1.0 1.0 1.2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ND 5.0 0.0 0.0 5.0 4.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Mean band intensity 1.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ND ND 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 2.0 ND NA NA NA NA NA NA

Lot 2 (n=5)

Product

Catalogue number Manufacturer

Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette 2.0 4.0 5.0 5.0 4.0 5.0 5.0 4.0 5.0 5.0 5.0 1.0 0.0 5.0 4.0 5.0 5.0 4.0 5.0 5.0 4.0 5.0 5.0 5.0 3.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 ND 1.0 1.0 1.0 1.0 1.0 1.0 1.2 1.0 1.2 1.0 1.0 1.0 5.0 4.0 0.0 5.0 5.0 5.0 5.0 2.0 5.0 5.0 5.0 5.0 5.0 5.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 1.0 ND 1.2 1.0 1.6 1.0 1.5 2.0 1.0 1.0 1.0 1.0 1.6 1.0 5.0 5.0 0.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 3.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.2 1.0 ND 1.0 1.0 1.0 1.0 1.0 2.0 1.0 1.0 1.0 1.0 1.8 1.0 NA NA NA NA NA NA 0.0 1.0 5.0 0.0 1.4 5.0 0.0 1.0 5.0 5.0 5.0 0.0 5.0 4.0 5.0 4.0 5.0 5.0 5.0 5.0 5.0 3.0 5.0 1.0 NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA 1.0 1.0 1.0 ND 1.0 1.0 1.4 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.8 1.0 NA NA NA NA NA NA 5.0 5.0 1.0 0.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 NA NA NA NA NA NA 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 1.0 1.0 1.0 2.0 2.0 1.0 2.0 1.0 1.0 5.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 1.0 2.0 2.0 1.0 2.0 1.0 1.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 1.0 2.0 1.0 1.0 2.0 1.0 1.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 4.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 1.0 2.0 1.6 1.0 2.0 1.0 1.0 0.0 3.0 5.0 5.0 5.0 5.0 5.0 5.0 3.0

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd.

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat RK MAL 001 Serums & Vaccines Ltd. ) I16FRC Premier Medical Corporation MALAG100 GenBody Inc. 20-0146-01 Green Cross Medical Science Corp. (Korea) AMAL-7025 Humasis Co., Ltd. ML03 ICT INTERNATIONAL A03-18-322 Artron Laboratories Inc. RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd NG-MAL-W23-001 SARL NG Biotech, Z.A. R0113C CTK Biotech Inc. 50310025 Zephyr Biomedicals IMPN-C52 Hangzhou Biotest Biotech Co. Ltd. 05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) Pvom, Plasmodium vivax, ovale and malariae

ND, not determined NA, not applicable Pf, Plasmodium falciparum 

Pv, Plasmodium vivax 

pan, Plasmodium species 

Annexes

121

12 2

Table A4.13: Heat stability testing results for P. falciparum (or pan) test line on parasite-negative samples. Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Baseline testing Manufacturer No. positive InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. SSA Diagnostics & Biotech Systems Premier Medical Corporation BIOSYNEX Shanghai Kehua Bio-engineering Co.,Ltd. Standard Diagnostics Inc. Vision Biotech (Pty) Ltd 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 1.0 2.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 4.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0

35 °C Lot 1 (n=4) No. positive No. invalid No. positive No. invalid No. positive Lot 2 (n=4) Lot 1 (n=4) No. invalid 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

45 °C Lot 2 (n=4) No. positive 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 2.0 2.0 4.0 0.0 0.0 0.0 0.0 0.0

Product No. invalid No. positive No. invalid

Catalogue number

Lot 1 (n=4) Lot 2 (n=4)

No. invalid 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

ITP11002TC1/TC40 IR016025 G0141 KMFC6001 I13FRC 0502_K25 KH-R-06-20 05FK50/05FK53 VB01

Pf only Advanced Quality™ One Step Malaria Pf Test Advantage P.f Malaria Card CareStart™Malaria HRP2 (Pf) diagnosticks- Malaria (Pf)Cassette WB First Response® Malaria Ag P. falciparum (HRP2) Card Test IMMUNOQUICK® MALARIA falciparum KHB® Malaria Ag P.f Rapid Test SD BIOLINE Malaria Antigen Pf Vision Malaria Pf Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums RK MAL 001 & Vaccines Ltd. ) I16FRC Premier Medical Corporation MALAG100 GenBody Inc. 20-0146-01 Green Cross Medical Science Corp. (Korea) AMAL-7025 Humasis Co., Ltd. ML03 ICT INTERNATIONAL A03-18-322 Artron Laboratories Inc. RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd NG-MAL-W23-001 SARL NG Biotech, Z.A. R0113C CTK Biotech Inc. 50310025 Zephyr Biomedicals IMPN-C52 Hangzhou Biotest Biotech Co. Ltd. 05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S

Table A4.13 (continued) Baseline testing 45 °C Lot 2 (n=4) No. positive 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

35 °C Lot 1 (n=4) No. positive 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Product No. positive ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. J. Mitra & Co. Pvt. Ltd. Access Bio. Inc. Pvom, Plasmodium vivax, ovale and malariae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Catalogue number Manufacturer No. invalid No. positive No. invalid No. invalid No. invalid No. positive No. invalid No. positive 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0

Lot 1 (n=4)

Lot 2 (n=4)

Lot 1 (n=4)

Lot 2 (n=4) No. invalid 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

AM4650-K

Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test Pan only Advantage Pan Malaria Card CareStart™Malaria pLDH (PAN)

GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

IR013025 G0111

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Pf, Plasmodium falciparum  a For pan-only tests

Pv, Plasmodium vivax 

pan, Plasmodium species 

Annexes

123

12 4

Table A4.13a: Heat stability testing results for pan test line of combination RDTs on parasite-negative samples. Positivity rate after 60 days at baseline (room temperature) and after incubation at 35 °C and 45 °C Baseline testing Manufacturer No. positive 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 NA NA NA NA NA NA

35 °C Lot 1 (n=4) No. positive No. invalid No. positive No. invalid No. positive Lot 2 (n=4) Lot 1 (n=4) No. invalid 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA

45 °C Lot 2 (n=4) No. positive 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 2.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA

Product No. invalid No. positive No. invalid

Catalogue number

Lot 1 (n=4) Lot 2 (n=4)

No. invalid 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NA NA NA NA NA NA

Pf and pan ACCUCARE ONE STEP MALARIA Pf/Pan Antigen Test Advanced Quality™ Rapid Malaria Test (Pf/Pan) Advantage Mal Card Advantage Malaria Pan + Pf Card BIOCREDIT Malaria Ag Pf/Pan (HRPII/pLDH) BioTracer™ Malaria Pf/PAN Rapid Card CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo Clearview® Malaria Dual DIAQUICK Malaria P.f/Pan Cassette

MAGC 25 ITP11005 IR221025 IR231025 C30RHA25 17012 G0131 VB20 Z11200CE

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013) ASAN Pharmaceutical Co., Ltd Genomix Molecular Diagnostics Pvt. Ltd. Bhat Bio-Tech India (P) Ltd. Blue Cross Bio-Medical (Beijing) Co., Ltd. Span Diagnostics Ltd. Guangzhou Wondfo Biotech Co. Ltd. Pvom, Plasmodium vivax, ovale and malariae

EzDx™ Malaria Pan/Pf Rapid Test Detection Kit

First Response® Malaria Ag. pLDH/HRP2 Combo Card Test GenBody™Malaria Pf/Pan Ag Genedia® Malaria P.f/Pan Ag Rapid Test Humasis Malaria P.f/Pan Antigen Test ICT Malaria Dual Test Malaria Pf./Pan Antigen (MAL Pf/Pan) Test Kit Malaria Pf/Pan One Step Rapid Test Malaria pf (pLDH) / PAN-pLDH Test Device MD Malaria Pf/Pan(pLDH) test NG-Test MALARIA Pf/Pan (pLDH) OnSite Pf/Pan Ag Rapid Test Parascreen® Rapid test for malaria Pan/Pf RightSign™ Malaria P.f./Pan Rapid Test Cassette SD BIOLINE Malaria Antigen Pf/Pan Vikia® Malaria Ag Pf/Pan Pf and Pv/Pvom ASAN Easy Test® Malaria Pf/Pan Ag Malaria Pf (HRPII)/ PV (PLDH) Antigen Detection Test Device Maleriscan® Malaria Pf/PAN (Pv, Pm, Po) 3 Line Antigen Test One Step Malaria P.F/P.V Test (Cassette) ParaHIT®fV Rapid test for P. falciparum and P. vivax Malaria - Device Wondfo® One Step Malaria P.f/P.v Whole Blood Test

LAB-CARE Diagnostics (India) PVT. LTD. InTec Products, Inc. J. Mitra & Co. Pvt. Ltd. J. Mitra & Co. Pvt. Ltd. RapiGEN INC. Bio Focus Co., Ltd. Access Bio. Inc. Orgenics Ltd.(IS) DIALAB GmbH Advy Chemical Pvt. Ltd. (Affiliate of Bharat Serums RK MAL 001 & Vaccines Ltd. ) I16FRC Premier Medical Corporation MALAG100 GenBody Inc. 20-0146-01 Green Cross Medical Science Corp. (Korea) AMAL-7025 Humasis Co., Ltd. ML03 ICT INTERNATIONAL A03-18-322 Artron Laboratories Inc. RT 20222 Zhejiang Orient Gene Biotech Co., Ltd. MFV-124 AZOG, INC. MDMALLDH001 Medical Diagnostech (Pty) Ltd NG-MAL-W23-001 SARL NG Biotech, Z.A. R0113C CTK Biotech Inc. 50310025 Zephyr Biomedicals IMPN-C52 Hangzhou Biotest Biotech Co. Ltd. 05FK60/05FK63 Standard Diagnostics Inc. 412499 IMACCESS S.A.S

AM4650-K GM006 MAT-PF/PAN-50 523352 55IC402-50 W056-C

NA, not applicable Pf, Plasmodium falciparum 

Pv, Plasmodium vivax 

pan, Plasmodium species 

Annex 5: Introducing RDT-based malaria diagnosis into national programmes Introduction of parasite-based diagnosis at small clinics and at village level for case management poses many challenges, not only of logistics but also in managing the health-seeking and health-providing behaviour of patients and health workers. These can be addressed by a clear, time-bound strategic plan covering planning, implementation, monitoring and evaluation of the diagnosis programme, which must begin well before RDTs are procured. Furthermore, funding for the programme must include a significant component for planning and coordination, sensitization, information, education and communication, training, quality assurance, monitoring, supervision and logistics, in addition to procurement. In the absence of such funding, much of the expenditure on RDTs will be wasted, and loss of confidence in RDT-based diagnosis can hinder strengthening of appropriate malaria case management. A focal person or persons should be available to coordinate the overall implementation plan and to ensure that the various agencies involved understand the process and their own roles. Examples of successful wide-scale introduction of malaria RDTs by various national programmes and comprehensive technical guidance on achieving universal access to malaria diagnostic testing have been reported (1,2). Figures A5.1 and A5.2 give examples of the steps and timelines for RDT implementation and budget components for a malaria diagnosis programme, respectively. These will have to be modified considerably for each programme.

Key challenges Changing past thinking that “fever equals malaria unless proven otherwise”. Introducing RDTs will disprove this statement. To have an impact on malaria diagnosis and treatment, RDTs must be seen to provide an accurate diagnosis by both health workers and patients, that is, they must be as good or better than those relied on previously. A health worker requires a good alternative to antimalarial medicines for the management of parasite-negative febrile patients. To achieve and maintain confidence in RDT-based diagnosis, a good quality assurance system must be in place. There must be satisfactory education of health workers and widespread community sensitization. Health workers should have understanding of other causes of fever in order to devise appropriate management algorithms for parasite-negative cases.

Changing and enforcing regulatory requirements At the national level, regulation might be required to control the importation and use of malaria RDTs, and new procedures for storage, distribution and inventory management, such as those used for medicines, might be necessary.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Annexes 125

12 6 Written MoH endorsement Written MoH endorsement Written MoH endorsement Dependent on registration process Determine site Commence testing

Figure A5.1. Example of malaria RDT implementation steps and timelinea

RDT IMPLEMENTATION TIMELINE

Coordinating group

Appoint malaria diagnosis coordinator(s)

Policy recommendations

Programme planning

Guidelinesb

Case management of fever of unknown origin

Case management of malaria

RDT (and microscopy) quality assurance

RDT transport and storage

Decide districts for initial / phased implementation

Fever management algorithm

Community sensitization

General health care providers’ education

Determine / designate transport and storage methods

Regulatory issues

Define collaborative roles (NMP and regulatory body)

Write/adopt regulatory guidleines

Create RDT registry for reference

Dsseminate regulatory criteria

Product selection, supply chain management

Select several products

Samples for ease-of-use assessment

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Final decision on RDT

Negotiate specifications with manufacturer

Competitive bidding and procurement

Receive first batch (of staggered delivery)

Distribution to field

Procure gloves

Procure sharps boxes

Procure other associated materials

RDT quality control

Write sentinel site SOP

Set up/engage field-based quality control monitoring sites

Decide on lot-testing site

Post-marketing surveillancec

Figure A6.1 (continued) May be conducted earlier, or already in place

Training

Conduct case management training for fever

Modify RDT instructions and training manual

Field-test modified training/instructions

Training of trainers and supervisors

Health worker training

Advocacy, communication, social mobilization

Engaging civil society organizations

Community sensitization

Engaging opinion leaders

General health care education

Monitoring and evaluation

Develop/adopt appropriate record forms

Define methods for capturing different indicators

Intergrate RDTs into the routine health information management system

Plan for a post-introduction programme review

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

MoH, ministry of health; NMP, national malaria programme a Adapted with permission from FIND and Uganda National Malaria Control Programme b May already be in place c Sentinel site mciroscopy, possibly positive control wells in future

Annexes

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Figure A5.2. Components of the budget for a malaria diagnosis programmea Component Activities specific to microscopy Activities specific to RDTs Activities for management of malaria and non-malaria fevers Fever management algorithm Other tests used at primary care level

Preparation of technical guidelines, standard operating procedures and checklists Guidelines Standard operating procedures for diagnostic testing Other standard operating procedures Training material Checklists for supervision Laboratory supervisionb Microscopy performance Proficiency testing, validation of routine slide results Training manual for microscopy Laboratory visitsb RDT transport and storage RDT performance RDT storage Training manual for RDTs Health facility visits Urine dipsticks, haemoglobin meter, haematocrit meter, glucometer Antibiotics, zinc, inhaled salbutamol, rehydration salts Training manuals for integrated management of fevers

Procurement and supply of commodities Diagnostic tests Medicines Other commodities Distribution of commodities to the field Quality management system Pre-shipment testing Training of focal people Monitoring the quality management system Training of health workers Training of tutors Training of health workers Training of supervisors Supervision Supervisory visits Laboratory visitsb Health facility visits Communication on other causes of fever Advocacy, communication and social mobilization Design of strategies and material Dissemination of key messages Monitoring and evaluation Updating the health information management system Train health workers in the new health information management system a Adapted b For

Microscopes and related supplies

RDT kits

Artemisinin-based combination therapy Gloves, lancets, alcohol, cotton-wool, timers, sharps boxes All items listed above

Lot-testing Quality management system for focal people Quality monitoring supervision visits and compilation of health information management data Tutors for RDT performance outside laboratories and clinical management of fever cases Health workers Clinical supervisors Clinicians

Expert microscopists Microscopists Laboratory supervisorsb

Communication on the need for malaria testing Through each delivery channel Add row for RDTs in laboratory report and column for malaria test results in clinicians’ book Training of person in charge or focal person for reporting on health information management in health facilities

Column for other test results in clinicians’ book

with permission (37) simplicity, activities specific to laboratories are listed under ‘Microscopy’, although both microscopy and RDT are generally performed in laboratories.

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References 1. Universal access to malaria diagnostic testing: an operational manual. Geneva: World Health Organization; 2011. 2. Thiam S, Thior M, Faye B, et al. Major reduction in anti-malarial drug consumption in Senegal after nation-wide introduction of malaria rapid diagnostic tests. PLoS One 2011; 6: e18419.

Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

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Malaria Rapid Diagnostic Test Performance – Results of WHO product testing of malaria RDTs: Round 5 (2013)

Global Malaria Programme World Health Organization 20, Avenue Appia 1211 Geneva 27 Switzerland infogmp@who.int www.who.int/malaria

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