10.07.2015 - altona Diagnostics GmbH - Letter to the editor/The Lancet
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).