- Email: [email protected]
ReEBOV Antigen Rapid Test kit for Ebola
Correspondence
www.thelancet.com Vol 386 December 5, 2015
support programmes should standardise charges on behalf of stakeholders (governments and private employers, especially agricultural firms, health insurance providers, etc) to mutualise the costs equitably. This crisis will not be overcome in laboratories or in universities, but in rural communities alongside health workers and victims of snake bites. We declare no competing interests.
*Jean-Philippe Chippaux, Achille Massougbodji, Amadou Diouf, Cellou M Baldé, Leslie V Boyer [email protected] UMR 216, Mother And Child Facing Tropical Diseases, Institut de Recherche pour le Développement, 08 BP 841, Cotonou, Benin (J-PC); Centre d’Etude et de Recherche sur le Paludisme Associé à la Grossesse et à l’Enfance, Faculté des Sciences de la Santé, Université d’Abomey-Calavi, Benin (AM); Centre Anti Poison du Sénégal, Ministère de la Santé et de la Prévention, Dakar, Senegal (AD); Laboratoire d’Entomologie Médicale et de Venimologie, Institut de Recherche et de Biologie Appliquée, Kindia, Guinea (CMB); and Venom Immunochemistry, Pharmacology and Emergency Response (VIPER) Institute, University of Arizona Health Sciences Center, Tucson, AZ, USA (LVB) 1
2
3
4
5
6
7
Doctors Without Borders. Global health community walks away from snakebite crisis as antivenom runs out. Sept 7, 2015. http://www.doctorswithoutborders.org/ article/global-health-community-walks-awaysnakebite-crisis-antivenom-runs-out (accessed Nov 9, 2015). Chippaux JP. Estimate of the burden of snakebites in sub-Saharan Africa: a meta-analytic approach. Toxicon 2011; 57: 586–99. WHO. Snake antivenom immunoglobulins. Geneva: World Health Organization, 2010. http://www.who.int/bloodproducts/snake_ antivenoms/snakeantivenomguide/en/ (accessed Nov 9, 2015). Stock RP, Massougbodji A, Alagón A, Chippaux JP. Bringing antivenoms to sub-Saharan Africa. Nat Biotechnol 2007; 25: 173–77. Chippaux JP, Massougbodji A, Stock RP, Alagon A, for the investigators of African Antivipmyn in Benin. Clinical trial of an F(ab’)2 polyvalent equine antivenom for African snake bites in Benin. Am J Trop Med Hyg 2007; 77: 538–46. Chippaux JP, Baldé MC, Sessinou É, Yéro Boiro M, Massougbodji A. Evaluation of a new polyvalent antivenom against snakebite envenomation (Inoserp® Panafricain) in two different epidemiological settings: northern Benin and Maritime Guinea. Med Sante Trop 2015; 25: 56–64 (in French). Chippaux JP. African Society of Toxinology: a new opportunity for integrating the control of envenomations in Africa. J Venom Anim Toxins Incl Trop Dis 2012; 18: 357–60.
ReEBOV Antigen Rapid Test kit for Ebola In their Article, Mara Jana Broadhurst and colleagues (Aug 29, p 867) 1 reported that the ReEBOV Antigen Rapid Test (Corgenix, Broomfield, CO, USA) had a sensitivity of 100% and a specificity of 92·2%. These results differ from those reported by the manufacturer 2 (62·1% sensitivity, 96·7% specificity), and from a study3 done in Sierra Leone by WHO (91·8%, 84·6%). These differences in sensitivity and specificity between studies might indicate selection bias. Broadhurst and colleagues recruited patients from a treatment centre who were found, on testing by RT-PCR, to have a high average viral load (maximum cycle threshold value <26). Additionally, patients with haemodynamic instability and patients who were unable to provide a specimen or give verbal consent were excluded from the study. The use of two reference methods for only a subset of patient samples further complicates interpretation, especially since one of the methods has not been independently assessed. Consequently, the ReEBOV Antigen Rapid Test seemed superior to one of the reference methods,4 despite the reference method having a detection limit more than 1000 times lower than that of the ReEBOV Antigen Rapid Test. WHO cautions that data from this study could be misinterpreted by users who assume that the ReEBOV Antigen Rapid Test will detect all cases, without considering the possibility of false negatives. We encourage more field studies be done to allow the research community and public health communities to compare data and draw sound conclusions that will help efforts to end Ebola virus disease and prevent, or better respond to, future epidemics.
Salah Malkawi/Stringer
region will be unable to deal with the 300 000 envenomings, 7000 deaths, and 10 000 permanent disabilities caused by snake bites annually.2 As pointed out by Sanofi-Pasteur, however, the production of Fav-Afrique was stopped not because of a halt in production, but because of a drastic fall in sales. By 2010, these sales had fallen to less than 1% of total annual burden of snake bites in sub-Saharan Africa—ie, about 3000 cases, resulting in 70 deaths.2 The antivenom shortage was also not caused by a technology deficit. Manufacturers have known how to make good antivenoms for decades. Several other antivenoms that meet the increasingly rigorous requirements of antivenom production exist. 3 Antivenoms should be effective against local species, safe enough for use in underequipped, peripheral health centres, physicochemically stable enough for shipping and storage in the African climate, and readily accessible to victims, who are usually people living in rural areas with few resources and little financial support.4 Since 2005, we have successfully used other antivenoms that are as effective and well tolerated as Fav-Afrique,5,6 and have the additional advantages of being lyophilised and costing Africans a third of the price of Fav-Afrique. These products neutralise the same venoms as Fav-Afrique and have been reformulated to optimise efficacy and safety. The solution to the crisis must address the root cause of the problem, which is a vicious circle that limits access to all antivenoms,4 not just Fav-Afrique. The shortage results from inadequate product distribution, training, and funding at the local level in Africa. The strategy recommended by the African Society of Toxinology,7 founded 3 years ago, addresses three basic points: better regionspecific antivenom development and distribution needs accurate epidemiological studies; medical staff need training to optimise product selection and use; and purchasing
We declare no competing interests. © 2015. World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved.
2253
Correspondence
Willy Urassa, Robyn Meurant, *David Wood [email protected] Department of Essential Medicine and Health Products, WHO, CH-1211 Geneva 27, Switzerland (WU, RM, DW) 1
2
3
4
Broadhurst MJ, Kelly JD, Miller A, et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet 2015; 386: 867–74. Corgenix. ReEBOV Antigen Rapid Test kit package insert. Feb 25, 2015. ftp://ftp. corgenix.com/Intra/ReEBOV/14005.pdf (accessed June 30, 2015). WHO. WHO emergency use assessment and listing for Ebola virus disease IVDs. Public report. Product: ReEBOV™ Antigen Rapid Test Kit. http://www.who.int/diagnostics_ laboratory/procurement/150219_reebov_ antigen_rapid_test_public_report.pdf (accessed June 30, 2015). WHO. WHO emergency quality assessment mechanism for Ebola virus disease IVDs. Public report. Product: RealStar Filovirus Screen RTPCR Kit 1.0. http://www.who.int/diagnostics_ laboratory/procurement/141125 _evd_public_report_altona_v1.pdf (accessed June 30, 2015).
Mara Jana Broadhurst and colleagues1 compared the ReEBOV Antigen Rapid Test with the RealStar Filovirus Screen RT-PCR kit 1.0 (altona Diagnostics, Hamburg, Germany) and their own real-time RT-PCR method, referred to as Trombley assay.2 We note major technical flaws in their study design. Particularly, that the RealStar Filovirus RT-PCR kit (altona assay), used as the reference diagnostic assay, was not used correctly. The SmartCycler II (Cepheid, Sunnyvale, CA, USA) used for DNA amplification and detection is not listed for use with the assay, neither in the user manual nor on the kit label.3 The 95% cutoff level of the assay (determined by probit analysis using in-vitro transcribed RNA) on the SmartCycler II is 40 copies per μL (95% CI 20–250), which is about 30 times higher than the limit of detection with the LightCycler 480 Instrument II (Roche Diagnostics, Mannheim, Germany). With the ABI Prism 7500 SDS instrument (ThermoFisher Scientific, Waltham, MA, USA), the authors detected in RNA cell culture supernatant at a dilution level of up to 2254
1:10⁸ using the altona assay. However, with the SmartCycler II, RNA could only be detected in supernatant at a dilution of up to 1:10⁵; a difference in sensitivity of at least two log units. The SmartCycler II was used in the study even though some of the authors had knowledge, through direct contact with the kit manufacturers, of the incompatibility of the SmartCycler II with the altona assay. To explain the discrepancy in sensitivity between ReEBOV Antigen Rapid Test and the altona assay, the authors refer to the latter as an imperfect reference. However, we would like to point out that the altona RealStar Filovirus RT-PCR kit 1.0 was not used according to our instructions. We are employees of altona Diagnostics.
*Stephan Ölschläger, Markus Heß [email protected] altona Diagnostics, 22767 Hamburg, Germany (SÖ, MH) 1
2
3
Broadhurst MJ, Kelly JD, Miller A, et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet 2015; 386: 867–74. Trombley AR, Wachter L, Garrison J, et al. Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses. Am J Trop Med Hyg 2010; 82: 954–60. altona Diagnostics. RealStar Filovirus Screen RT-PCR Kit 1.0, instructions for use. August, 2014. http://www.altona-diagnostics.com/tl_ files/website/downloads/RealStar_ Filovirus%20Screen%20RT-PCR%20Kit%20 10_CE_WEB_2014-08-29.pdf (accessed Nov 10, 2015).
In view of the ongoing outbreak of Ebola virus in west Africa and the scarce laboratory facilities in Ebola epidemic areas, development and assessment of rapid, point-of-care tests for Ebola virus disease is needed. The ReEBOV Antigen Rapid Test investigated by Mara Jana Broadhurst and colleagues 1 meets these expectations. As underlined by the authors, we showed that the reference RT-PCR assay used in their study (RealStar Filovirus RT-PCR kit 1.02) was not optimum and overestimated the sensitivity of the ReEBOV
Antigen Rapid Test. 3 In a field study, we tested RealStar Filovirus RT-PCR kit 1.0 (altona assay) versus Lightmix Modular Ebola Virus Zaire (TIBMOLBIOL, Berlin, Germany4) with the same RNA extraction technique and RT-PCR platform as Broadhurst and colleagues. The cycle threshold values were similar to the RT-PCR methods we tested for high viral load (cycle threshold value <30), and we confirm a drop-off in the sensitivity of the altona assay for cycle threshold values greater than 30. We also showed that this low sensitivity did not seem to be related to RNA extraction or amplification methods, as suggested by Broadhurst and colleagues. A broad range of virus targets could account for this reduced sensitivity. The RealStar Filovirus RT-PCR kit 1.0 is designed to detect Ebola virus and Marburg virus specifically, whereas other RT-PCR tests (such as the Trombley assay5 and RealStar Ebolavirus RT-PCR kit 1.06) are specific to Ebola virus only. We suggest that the sensitivity of the ReEBOV Antigen Rapid Test reported in this Article, or in future studies, should be assessed with a more efficient RT-PCR assay for all negative samples or samples for which the cycle threshold value is greater than 30. However, our field experience confirmed that in samples from patients admitted to Ebola treatment centres, cycle threshold values from rapid diagnostic tests are frequently less than 30 (93% of cases),7 and this type of rapid diagnostic test is essential in disease outbreak conditions. We declare no competing interests.
*Frédéric Janvier, Emmanuel Sagui, Vincent Foissaud [email protected] Healthcare Workers Ebola Treatment Center, Conakry, Guinea (FJ, ES, VF) 1
Broadhurst MJ, Kelly JD, Miller A, et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet 2015; 386: 867–74.
www.thelancet.com Vol 386 December 5, 2015
www.thelancet.com Vol 386 December 5, 2015
support programmes should standardise charges on behalf of stakeholders (governments and private employers, especially agricultural firms, health insurance providers, etc) to mutualise the costs equitably. This crisis will not be overcome in laboratories or in universities, but in rural communities alongside health workers and victims of snake bites. We declare no competing interests.
*Jean-Philippe Chippaux, Achille Massougbodji, Amadou Diouf, Cellou M Baldé, Leslie V Boyer [email protected] UMR 216, Mother And Child Facing Tropical Diseases, Institut de Recherche pour le Développement, 08 BP 841, Cotonou, Benin (J-PC); Centre d’Etude et de Recherche sur le Paludisme Associé à la Grossesse et à l’Enfance, Faculté des Sciences de la Santé, Université d’Abomey-Calavi, Benin (AM); Centre Anti Poison du Sénégal, Ministère de la Santé et de la Prévention, Dakar, Senegal (AD); Laboratoire d’Entomologie Médicale et de Venimologie, Institut de Recherche et de Biologie Appliquée, Kindia, Guinea (CMB); and Venom Immunochemistry, Pharmacology and Emergency Response (VIPER) Institute, University of Arizona Health Sciences Center, Tucson, AZ, USA (LVB) 1
2
3
4
5
6
7
Doctors Without Borders. Global health community walks away from snakebite crisis as antivenom runs out. Sept 7, 2015. http://www.doctorswithoutborders.org/ article/global-health-community-walks-awaysnakebite-crisis-antivenom-runs-out (accessed Nov 9, 2015). Chippaux JP. Estimate of the burden of snakebites in sub-Saharan Africa: a meta-analytic approach. Toxicon 2011; 57: 586–99. WHO. Snake antivenom immunoglobulins. Geneva: World Health Organization, 2010. http://www.who.int/bloodproducts/snake_ antivenoms/snakeantivenomguide/en/ (accessed Nov 9, 2015). Stock RP, Massougbodji A, Alagón A, Chippaux JP. Bringing antivenoms to sub-Saharan Africa. Nat Biotechnol 2007; 25: 173–77. Chippaux JP, Massougbodji A, Stock RP, Alagon A, for the investigators of African Antivipmyn in Benin. Clinical trial of an F(ab’)2 polyvalent equine antivenom for African snake bites in Benin. Am J Trop Med Hyg 2007; 77: 538–46. Chippaux JP, Baldé MC, Sessinou É, Yéro Boiro M, Massougbodji A. Evaluation of a new polyvalent antivenom against snakebite envenomation (Inoserp® Panafricain) in two different epidemiological settings: northern Benin and Maritime Guinea. Med Sante Trop 2015; 25: 56–64 (in French). Chippaux JP. African Society of Toxinology: a new opportunity for integrating the control of envenomations in Africa. J Venom Anim Toxins Incl Trop Dis 2012; 18: 357–60.
ReEBOV Antigen Rapid Test kit for Ebola In their Article, Mara Jana Broadhurst and colleagues (Aug 29, p 867) 1 reported that the ReEBOV Antigen Rapid Test (Corgenix, Broomfield, CO, USA) had a sensitivity of 100% and a specificity of 92·2%. These results differ from those reported by the manufacturer 2 (62·1% sensitivity, 96·7% specificity), and from a study3 done in Sierra Leone by WHO (91·8%, 84·6%). These differences in sensitivity and specificity between studies might indicate selection bias. Broadhurst and colleagues recruited patients from a treatment centre who were found, on testing by RT-PCR, to have a high average viral load (maximum cycle threshold value <26). Additionally, patients with haemodynamic instability and patients who were unable to provide a specimen or give verbal consent were excluded from the study. The use of two reference methods for only a subset of patient samples further complicates interpretation, especially since one of the methods has not been independently assessed. Consequently, the ReEBOV Antigen Rapid Test seemed superior to one of the reference methods,4 despite the reference method having a detection limit more than 1000 times lower than that of the ReEBOV Antigen Rapid Test. WHO cautions that data from this study could be misinterpreted by users who assume that the ReEBOV Antigen Rapid Test will detect all cases, without considering the possibility of false negatives. We encourage more field studies be done to allow the research community and public health communities to compare data and draw sound conclusions that will help efforts to end Ebola virus disease and prevent, or better respond to, future epidemics.
Salah Malkawi/Stringer
region will be unable to deal with the 300 000 envenomings, 7000 deaths, and 10 000 permanent disabilities caused by snake bites annually.2 As pointed out by Sanofi-Pasteur, however, the production of Fav-Afrique was stopped not because of a halt in production, but because of a drastic fall in sales. By 2010, these sales had fallen to less than 1% of total annual burden of snake bites in sub-Saharan Africa—ie, about 3000 cases, resulting in 70 deaths.2 The antivenom shortage was also not caused by a technology deficit. Manufacturers have known how to make good antivenoms for decades. Several other antivenoms that meet the increasingly rigorous requirements of antivenom production exist. 3 Antivenoms should be effective against local species, safe enough for use in underequipped, peripheral health centres, physicochemically stable enough for shipping and storage in the African climate, and readily accessible to victims, who are usually people living in rural areas with few resources and little financial support.4 Since 2005, we have successfully used other antivenoms that are as effective and well tolerated as Fav-Afrique,5,6 and have the additional advantages of being lyophilised and costing Africans a third of the price of Fav-Afrique. These products neutralise the same venoms as Fav-Afrique and have been reformulated to optimise efficacy and safety. The solution to the crisis must address the root cause of the problem, which is a vicious circle that limits access to all antivenoms,4 not just Fav-Afrique. The shortage results from inadequate product distribution, training, and funding at the local level in Africa. The strategy recommended by the African Society of Toxinology,7 founded 3 years ago, addresses three basic points: better regionspecific antivenom development and distribution needs accurate epidemiological studies; medical staff need training to optimise product selection and use; and purchasing
We declare no competing interests. © 2015. World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved.
2253
Correspondence
Willy Urassa, Robyn Meurant, *David Wood [email protected] Department of Essential Medicine and Health Products, WHO, CH-1211 Geneva 27, Switzerland (WU, RM, DW) 1
2
3
4
Broadhurst MJ, Kelly JD, Miller A, et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet 2015; 386: 867–74. Corgenix. ReEBOV Antigen Rapid Test kit package insert. Feb 25, 2015. ftp://ftp. corgenix.com/Intra/ReEBOV/14005.pdf (accessed June 30, 2015). WHO. WHO emergency use assessment and listing for Ebola virus disease IVDs. Public report. Product: ReEBOV™ Antigen Rapid Test Kit. http://www.who.int/diagnostics_ laboratory/procurement/150219_reebov_ antigen_rapid_test_public_report.pdf (accessed June 30, 2015). WHO. WHO emergency quality assessment mechanism for Ebola virus disease IVDs. Public report. Product: RealStar Filovirus Screen RTPCR Kit 1.0. http://www.who.int/diagnostics_ laboratory/procurement/141125 _evd_public_report_altona_v1.pdf (accessed June 30, 2015).
Mara Jana Broadhurst and colleagues1 compared the ReEBOV Antigen Rapid Test with the RealStar Filovirus Screen RT-PCR kit 1.0 (altona Diagnostics, Hamburg, Germany) and their own real-time RT-PCR method, referred to as Trombley assay.2 We note major technical flaws in their study design. Particularly, that the RealStar Filovirus RT-PCR kit (altona assay), used as the reference diagnostic assay, was not used correctly. The SmartCycler II (Cepheid, Sunnyvale, CA, USA) used for DNA amplification and detection is not listed for use with the assay, neither in the user manual nor on the kit label.3 The 95% cutoff level of the assay (determined by probit analysis using in-vitro transcribed RNA) on the SmartCycler II is 40 copies per μL (95% CI 20–250), which is about 30 times higher than the limit of detection with the LightCycler 480 Instrument II (Roche Diagnostics, Mannheim, Germany). With the ABI Prism 7500 SDS instrument (ThermoFisher Scientific, Waltham, MA, USA), the authors detected in RNA cell culture supernatant at a dilution level of up to 2254
1:10⁸ using the altona assay. However, with the SmartCycler II, RNA could only be detected in supernatant at a dilution of up to 1:10⁵; a difference in sensitivity of at least two log units. The SmartCycler II was used in the study even though some of the authors had knowledge, through direct contact with the kit manufacturers, of the incompatibility of the SmartCycler II with the altona assay. To explain the discrepancy in sensitivity between ReEBOV Antigen Rapid Test and the altona assay, the authors refer to the latter as an imperfect reference. However, we would like to point out that the altona RealStar Filovirus RT-PCR kit 1.0 was not used according to our instructions. We are employees of altona Diagnostics.
*Stephan Ölschläger, Markus Heß [email protected] altona Diagnostics, 22767 Hamburg, Germany (SÖ, MH) 1
2
3
Broadhurst MJ, Kelly JD, Miller A, et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet 2015; 386: 867–74. Trombley AR, Wachter L, Garrison J, et al. Comprehensive panel of real-time TaqMan polymerase chain reaction assays for detection and absolute quantification of filoviruses, arenaviruses, and New World hantaviruses. Am J Trop Med Hyg 2010; 82: 954–60. altona Diagnostics. RealStar Filovirus Screen RT-PCR Kit 1.0, instructions for use. August, 2014. http://www.altona-diagnostics.com/tl_ files/website/downloads/RealStar_ Filovirus%20Screen%20RT-PCR%20Kit%20 10_CE_WEB_2014-08-29.pdf (accessed Nov 10, 2015).
In view of the ongoing outbreak of Ebola virus in west Africa and the scarce laboratory facilities in Ebola epidemic areas, development and assessment of rapid, point-of-care tests for Ebola virus disease is needed. The ReEBOV Antigen Rapid Test investigated by Mara Jana Broadhurst and colleagues 1 meets these expectations. As underlined by the authors, we showed that the reference RT-PCR assay used in their study (RealStar Filovirus RT-PCR kit 1.02) was not optimum and overestimated the sensitivity of the ReEBOV
Antigen Rapid Test. 3 In a field study, we tested RealStar Filovirus RT-PCR kit 1.0 (altona assay) versus Lightmix Modular Ebola Virus Zaire (TIBMOLBIOL, Berlin, Germany4) with the same RNA extraction technique and RT-PCR platform as Broadhurst and colleagues. The cycle threshold values were similar to the RT-PCR methods we tested for high viral load (cycle threshold value <30), and we confirm a drop-off in the sensitivity of the altona assay for cycle threshold values greater than 30. We also showed that this low sensitivity did not seem to be related to RNA extraction or amplification methods, as suggested by Broadhurst and colleagues. A broad range of virus targets could account for this reduced sensitivity. The RealStar Filovirus RT-PCR kit 1.0 is designed to detect Ebola virus and Marburg virus specifically, whereas other RT-PCR tests (such as the Trombley assay5 and RealStar Ebolavirus RT-PCR kit 1.06) are specific to Ebola virus only. We suggest that the sensitivity of the ReEBOV Antigen Rapid Test reported in this Article, or in future studies, should be assessed with a more efficient RT-PCR assay for all negative samples or samples for which the cycle threshold value is greater than 30. However, our field experience confirmed that in samples from patients admitted to Ebola treatment centres, cycle threshold values from rapid diagnostic tests are frequently less than 30 (93% of cases),7 and this type of rapid diagnostic test is essential in disease outbreak conditions. We declare no competing interests.
*Frédéric Janvier, Emmanuel Sagui, Vincent Foissaud [email protected] Healthcare Workers Ebola Treatment Center, Conakry, Guinea (FJ, ES, VF) 1
Broadhurst MJ, Kelly JD, Miller A, et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. Lancet 2015; 386: 867–74.
www.thelancet.com Vol 386 December 5, 2015