We would like to comment on the Article by Broadhurst et al.1 The authors compared the ReEBOV Antigen Rapid Test Kit (Corgenix Inc) with the RealStar® Filovirus Screen RT-PCR Kit 1.0 (altona Diagnostics GmbH) and an in-house developed real-time RT-PCR method referred to as Trombley-assay.2
We see major technical weaknesses in the study design. In particular, that the RealStar® Filovirus RT-PCR Kit 1.0, as the reference diagnostic assay, was not used properly. The SmartCycler® II (Cepheid) was used for amplification and detection, an instrument not listed to be used with this kit, neither in the user manual nor on the kit label. The 95% cut off level of the assay (determined with in-vitro transcribed RNA) on the SmartCycler® II is 40 copies/µl (95% confidence interval: 20-250 copies/µl). This is approximately 30 times higher than the limit of detection on the LightCycler® 480 Instrument II (Roche). On the ABI Prism® 7500 SDS Instrument (Applied Biosystems), the RealStar® Filovirus RT-PCR Kit 1.0 Kit detected diluted RNA from cell-culture supernatant up to a dilution level of 1:10e+08. On the SmartCycler® II, the same samples were positive only up to a dilution of 1:10e+05. This reflects a difference in sensitivity of at least two log units. Despite the fact that some of the authors had knowledge of the inadequateness of the SmartCycler® II in conjunction with the RealStar® Filovirus Screen RT-PCR Kit 1.0 beforehand, via direct contact with altona Diagnostics, the SmartCycler® II was used in the study.
The authors explain the discrepancy between ReEBOV and the RealStar® Filovirus Screen RT-PCR Kit 1.0 with the latter being an “imperfect reference”. However, we would like to point out that the RealStar® Filovirus RT-PCR Kit 1.0 Kit was not used according to our manufacturer’s instructions in this study.
1 Broadhurst, M. J. et al. ReEBOV Antigen Rapid Test kit for point-of-care and laboratory-based testing for Ebola virus disease: a field validation study. The Lancet doi:10.1016/S0140-6736(15)61042-X
2 Trombley, A. R. et al. Comprehensive Panel of Real-Time TaqManTM Polymerase Chain Reaction Assays for Detection and Absolute Quantification of Filoviruses, Arenaviruses, and New World Hantaviruses. Am. J. Trop. Med. Hyg. 82, 954–960 (2010).
altona Diagnostics GmbH Letter to the editor_The Lancet_2015-07-10pdf