- Email: [email protected]
ReEBOV Antigen Rapid Test kit for Ebola
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
Correspondence
3
4
5
6
7
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%2010_ CE_WEB_2014-08-29.pdf (accessed Nov 9, 2015). Janvier F, Gorbatch S, Queval L, et al. Difficulties of interpretation of Zaire Ebola Virus PCR results and implication in the field. J Clin Virol 2015; 67: 36–37. TIB MOLBIOL. LightMix Modular Ebola Virus Zaire (2014), instructions for use. April, 2014. http://www.roche-as.es/logs/MDx_53–0649– 96_Ebola_V140404_07383428001.pdf (accessed Nov 9, 2015). 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 Ebolavirus RT-PCR Kit 1.0, instructions for use. November, 2014. http://www.altona-diagnostics.com/tl_files/ website/downloads/RealStar_Ebolavirus%20 1.0%20RT-PCR%20Kit%20Instructions%20 for%20use%20clean%202014-11-26.pdf (accessed Nov 9, 2015). Janvier F, Foissaud V, Sagui E, et al. Monitoring of laboratory values over the course of Ebola virus disease in Healthcare Workers Ebola Treatment Center, Conakry, Guinea. J Infect Dis (in press).
Simple, cheap, and accurate rapid diagnostic tests for Ebola virus are urgently needed. The important study by Mara Jana Broadhurst and colleagues1 highlights the challenges of assessing such tests on whole blood when the number of positive patients is small and no agreed reference standard exists. These factors could explain the increased sensitivity of the ReEBOV Antigen Rapid Test compared with previous data from WHO.2 Although the authors note the limitations of their findings, we urge readers to be cautious with two of the conclusions. First, the authors suggest that a test with imperfect sensitivity and specificity would assist in triage of patients with high viral loads. However, this approach is only possible in specialised facilities in which patients with false-positive test results can be protected from nosocomial infection and patients with false-negative results can be safely isolated until confirmatory PCR results are obtained. In addition to the consequences for individuals, news of www.thelancet.com Vol 386 December 5, 2015
false test results can be disastrous at the community level, with potentially serious consequences for outbreak control.3 Second, the authors state that rapid diagnostic tests for Ebola virus are easy to use; however, their use necessitates strict biosafety protocols and equipment to avoid health-care workers becoming infected.4 In view of these limitations, the ReEBOV Antigen Rapid Test can be safely used only in a setting with strict biosafety measures. Thus, the kit confers little added value since PCR technologies are already available in such settings. Technical guidance on rapid diagnostic tests for Ebola virus is urgently needed and should include a description of how test results should be incorporated in diagnostic algorithms, taking into account the availability of new, integrated PCR methods5,6 and biosafety measures. We declare no competing interests.
*Erwan Piriou, Arlene C Chua, Armand G Sprecher [email protected] Médecins Sans Frontières, 1018 DD Amsterdam, Netherlands (EP); Médecins Sans Frontières, Access Campaign, Geneva, Switzerland (ACC); Department of Infectious Diseases, Institute of Infectious Diseases and Epidemiology, Singapore (ACC); and Médecins Sans Frontières, Brussels, Belgium (AGS) 1
2
3
4
5
6
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 March 9, 2015). The Lancet. Ebola in west Africa: gaining community trust and confidence. Lancet 2014; 383: 1946. WHO. Health worker Ebola infections in Guinea, Liberia and Sierra Leone: a preliminary report. May 21, 2015. http://apps.who.int/iris/ bitstream/10665/171823/1/WHO_EVD_SDS_ REPORT_2015·1_eng.pdf?ua=1 (accessed July 8, 2015). FIND. Situational review of Ebola diagnostics and opportunities for rapid improvement. Nov 21, 2014. http://www.finddiagnostics. org/export/sites/default/resource-centre/ news/pdf/ebola_diagnostics_summary_v12. pdf (accessed Nov 9, 2015). FDA. 2014 Ebola virus emergency use authorizations. May 13, 2015. http://www.fda.gov/MedicalDevices/Safety/ EmergencySituations/ucm161496.htm#ebola (accessed Nov 9, 2015).
Authors’ reply We appreciate the responses to our Article,1 which, to date, is one of the few independent field studies of Ebola diagnostic tests during the 2014–15 outbreak. In response to Willy Urassa and colleagues, we reject the assertion of significant selection bias in our study. As a field study, patient selection was dictated by presentation of patients to Ebola care centres. In our study population, the duration of symptoms at the time of presentation was representative of our clinical experience throughout several months of the outbreak and comparable to that reported from another centre.2 Whereas the maximum cycle threshold value, as measured by the altona RealStar Filovirus Screen RT-PCR kit 1.0, was 26·3 in our study population, our Trombley assay data indicated that our study population included patients whose blood samples yielded higher cycle threshold values. Although we had predefined study exclusion criteria, we did not need to exclude any patients on the basis of haemodynamic instability, inability to cooperate with sample collection, or inability to provide consent. Our Article clearly acknowledges the limitations of our study, including the use of a reference standard that had imperfect sensitivity and our inability to retest all samples with the Trombley assay. We understand the concern that our data could be misinterpreted if readers do not note these limitations, but we went to great lengths to articulate them. Finally, we also clearly acknowledge the differences between our results and those of the WHO study,3 for which little methodological detail is publicly available for comparison. Of note, the WHO study was restricted to laboratory-based testing of frozen plasma and fresh venous whole blood specimens—ie, no point-of-care testing was done. In response to Stephan Ölschläger and Markus Heß, we acknowledge that
Jerrold Diederich/US Army Africa/Science Photo Library
2
2255
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
Correspondence
3
4
5
6
7
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%2010_ CE_WEB_2014-08-29.pdf (accessed Nov 9, 2015). Janvier F, Gorbatch S, Queval L, et al. Difficulties of interpretation of Zaire Ebola Virus PCR results and implication in the field. J Clin Virol 2015; 67: 36–37. TIB MOLBIOL. LightMix Modular Ebola Virus Zaire (2014), instructions for use. April, 2014. http://www.roche-as.es/logs/MDx_53–0649– 96_Ebola_V140404_07383428001.pdf (accessed Nov 9, 2015). 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 Ebolavirus RT-PCR Kit 1.0, instructions for use. November, 2014. http://www.altona-diagnostics.com/tl_files/ website/downloads/RealStar_Ebolavirus%20 1.0%20RT-PCR%20Kit%20Instructions%20 for%20use%20clean%202014-11-26.pdf (accessed Nov 9, 2015). Janvier F, Foissaud V, Sagui E, et al. Monitoring of laboratory values over the course of Ebola virus disease in Healthcare Workers Ebola Treatment Center, Conakry, Guinea. J Infect Dis (in press).
Simple, cheap, and accurate rapid diagnostic tests for Ebola virus are urgently needed. The important study by Mara Jana Broadhurst and colleagues1 highlights the challenges of assessing such tests on whole blood when the number of positive patients is small and no agreed reference standard exists. These factors could explain the increased sensitivity of the ReEBOV Antigen Rapid Test compared with previous data from WHO.2 Although the authors note the limitations of their findings, we urge readers to be cautious with two of the conclusions. First, the authors suggest that a test with imperfect sensitivity and specificity would assist in triage of patients with high viral loads. However, this approach is only possible in specialised facilities in which patients with false-positive test results can be protected from nosocomial infection and patients with false-negative results can be safely isolated until confirmatory PCR results are obtained. In addition to the consequences for individuals, news of www.thelancet.com Vol 386 December 5, 2015
false test results can be disastrous at the community level, with potentially serious consequences for outbreak control.3 Second, the authors state that rapid diagnostic tests for Ebola virus are easy to use; however, their use necessitates strict biosafety protocols and equipment to avoid health-care workers becoming infected.4 In view of these limitations, the ReEBOV Antigen Rapid Test can be safely used only in a setting with strict biosafety measures. Thus, the kit confers little added value since PCR technologies are already available in such settings. Technical guidance on rapid diagnostic tests for Ebola virus is urgently needed and should include a description of how test results should be incorporated in diagnostic algorithms, taking into account the availability of new, integrated PCR methods5,6 and biosafety measures. We declare no competing interests.
*Erwan Piriou, Arlene C Chua, Armand G Sprecher [email protected] Médecins Sans Frontières, 1018 DD Amsterdam, Netherlands (EP); Médecins Sans Frontières, Access Campaign, Geneva, Switzerland (ACC); Department of Infectious Diseases, Institute of Infectious Diseases and Epidemiology, Singapore (ACC); and Médecins Sans Frontières, Brussels, Belgium (AGS) 1
2
3
4
5
6
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 March 9, 2015). The Lancet. Ebola in west Africa: gaining community trust and confidence. Lancet 2014; 383: 1946. WHO. Health worker Ebola infections in Guinea, Liberia and Sierra Leone: a preliminary report. May 21, 2015. http://apps.who.int/iris/ bitstream/10665/171823/1/WHO_EVD_SDS_ REPORT_2015·1_eng.pdf?ua=1 (accessed July 8, 2015). FIND. Situational review of Ebola diagnostics and opportunities for rapid improvement. Nov 21, 2014. http://www.finddiagnostics. org/export/sites/default/resource-centre/ news/pdf/ebola_diagnostics_summary_v12. pdf (accessed Nov 9, 2015). FDA. 2014 Ebola virus emergency use authorizations. May 13, 2015. http://www.fda.gov/MedicalDevices/Safety/ EmergencySituations/ucm161496.htm#ebola (accessed Nov 9, 2015).
Authors’ reply We appreciate the responses to our Article,1 which, to date, is one of the few independent field studies of Ebola diagnostic tests during the 2014–15 outbreak. In response to Willy Urassa and colleagues, we reject the assertion of significant selection bias in our study. As a field study, patient selection was dictated by presentation of patients to Ebola care centres. In our study population, the duration of symptoms at the time of presentation was representative of our clinical experience throughout several months of the outbreak and comparable to that reported from another centre.2 Whereas the maximum cycle threshold value, as measured by the altona RealStar Filovirus Screen RT-PCR kit 1.0, was 26·3 in our study population, our Trombley assay data indicated that our study population included patients whose blood samples yielded higher cycle threshold values. Although we had predefined study exclusion criteria, we did not need to exclude any patients on the basis of haemodynamic instability, inability to cooperate with sample collection, or inability to provide consent. Our Article clearly acknowledges the limitations of our study, including the use of a reference standard that had imperfect sensitivity and our inability to retest all samples with the Trombley assay. We understand the concern that our data could be misinterpreted if readers do not note these limitations, but we went to great lengths to articulate them. Finally, we also clearly acknowledge the differences between our results and those of the WHO study,3 for which little methodological detail is publicly available for comparison. Of note, the WHO study was restricted to laboratory-based testing of frozen plasma and fresh venous whole blood specimens—ie, no point-of-care testing was done. In response to Stephan Ölschläger and Markus Heß, we acknowledge that
Jerrold Diederich/US Army Africa/Science Photo Library
2
2255