1 DISCLAIMER This paper was submitted to the Bulletin of the World Health Organization and was posted to the Zika open site, according to the protocol for public health emergencies for international concern as described in Christopher Dye et al. (http://dx.doi.org/10.2471/BLT.16.170860). The information herein is available for unrestricted use, distribution and reproduction in any medium, provided that the original work is properly cited as indicated by the Creative Commons Attribution 3.0 Intergovernmental Organizations licence (CC BY IGO 3.0). RECOMMENDED CITATION Corman VM, Rasche A, Baronti C, Aldabbagh S, Cadar D, Reusken CBEM et al. Clinical comparison, standardization and optimization of Zika virus molecular detection [Submitted]. Bull World Health Organ E-pub: 19 Apr 2016. doi: http://dx.doi.org/10.2471/BLT.16.175950 . Clinical comparison, standardization and optimization of Zika virus molecular detection Victor M. Corman, a Andrea Rasche, a Cecile Baronti, b Souhaib Aldabbagh, a Daniel Cadar, c Chantal B.E.M. Reusken, d Suzan D. Pas, d Abraham Goorhuis, e Janke Schinkel, f Richard Molenkamp, f Beate M. Kuemmerer, a Tobias Bleicker, a Sebastian Brünink,a Monika Eschbach- Bludau, a Anna M. Eis-Hübinger, a Marion P. Koopmans, d Jonas Schmidt- Chanasit, c Martin P. Grobusch, e Xavier de Lamballerie, b Christian Drosten a & Jan Felix Drexler a aInstitute of Virology, University of Bonn Medical Centre, Bonn 53127, Germany bAix Marseille Université, IRD French Institute of Research for Development, EHESP French School of Public Health, EPV UMR_D 190 "Emergence des Pathologies Virales", France cBernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Hamburg, Germany dErasmus MC, Department of Viroscience, 3000 CA, Rotterdam, the Netherlands eCenter of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam, the Netherlands fClinical Virology Laboratory, Department of Medical Microbiology, Academic Medical Center, Amsterdam, the Netherlands (JS, RM) Correspondence to Jan Felix Drexler (e-mail: [email protected] ) (Submitted: 18 April 2016 – Published online: 19 April 2016) 2 One sentence summary: Very low Zika virus loads in blood and urine from patients and low sensitivity of several published assays imply invalid test results during the current outbreak and demand highly accurate diagnostics. What was already known about the topic concerned: The Zika virus (ZIKV) has been known since the 1950ies. There are six published real time RT-PCR-based protocols (qPCR), several of which are widely used for virus detection in the context of the current outbreak in the Americas. Data on analytical sensitivity and compatibility with current ZIKV outbreak strains is not available for most of these assays and the comparability of these assays between laboratories remains unknown. What new knowledge the manuscript contributes: Several assays may be of limited utility for patient diagnostics during the current outbreak because of low sensitivity and incompatibility with ZIKV outbreak strains. ZIKV loads were low irrespective of sample type, implying patients may go undiagnosed during the current outbreak due to limited assay sensitivity. The novel control RNA generated in this study allowed uniform ZIKV quantification and will prove useful for patient characterization in multicentric studies on ZIKV pathogenesis. 3 Abstract Objective Molecular Zika virus (ZIKV) detection is key to patient diagnostics during the current outbreak. Here, we address standardization and diagnostic performance of widely used real- time RT-PCR (qPCR) protocols for ZIKV detection. Methods Two novel qPCR protocols covering the currently known ZIKV genetic variability were analyzed together with all six published qPCR protocols. The performance of all assays was compared using a newly constructed universal control RNA (ucRNA) that contains the target regions of all compared assays on one strand of synthetic RNA. Findings Up to 10 oligonucleotide mismatches with ZIKV outbreak strains existed in published qPCR protocols. The analytical sensitivity of most assays was around 5 copies per reaction, whereas three assays showed a 3-250-fold decreased sensitivity. The novel ucRNA enabled uniform ZIKV quantification, whereas comparisons of PCR threshold cycles (C T-values) resulted in up to 20-fold misquantification between protocols. Mean ZIKV loads in 33 outbreak samples were 10 4 RNA copies/mL of blood (range; 10 2-4x10 5) and 5x10 3 RNA copies/ mL of urine (range; 4x10 2-5.9x10 4) within two weeks after symptom onset. Conclusion Several ZIKV qPCR protocols show limited sensitivity and incompatibility with ZIKV outbreak strains. ZIKV infection results in low virus concentrations close to the technical limit of detection irrespective of sample type, implying that 20%-80% of patients may go undiagnosed due to limited sensitivity of molecular tests. We provide updated protocols for ZIKV detection that are suitable for all ZIKV strains. The ucRNA will enable coordinated implementation of ZIKV molecular diagnostics across regions and within multicentric clinical trials. 4 Introduction The arthropod-borne Zika virus (ZIKV, genus Flavivirus , family Flaviviridae ) was first identified in 1947 in Uganda (1). Only sporadic human cases were reported prior to the 2007 outbreak in the Micronesian Yap islands from which 49 confirmed and 59 probable cases were reported (2, 3). ZIKV infections are frequently asymptomatic or show only mild clinical symptoms, including fever, arthralgia and rash (2, 4). However, severe neurological complications, including the Guillain-Barré syndrome were reported from previous outbreaks (5, 6). The current outbreak is additionally associated with fetal malformations (7-10). In Latin America and the Caribbean, ZIKV infection cannot be reliably diagnosed by clinical presentation because the co-circulating dengue virus (DENV) and chikungunya virus (CHIKV) cause similar symptoms. Serology is challenging because of the cross-reactivity of antibodies caused by endemic flaviviruses including DENV, St Louis encephalitis, and West Nile virus (WNV) (4, 11, 12). Reliable detection of ZIKV is key to investigations of ZIKV epidemiology and pathogenesis (13). Because of potential association with neurological fetopathies, ZIKV infection should ideally be diagnosed already in the first trimester of pregnancy when neurological development takes place (14). Direct detection of ZIKV is also key to investigations of alternative transmission routes such as semen and blood donations. There are six widely used real-time RT-PCR assays for ZIKV detection (11, 15-17). An additional novel real-time RT-PCR assay has been recommended by the Pan American health association (PAHO) for the current outbreak (13). It is unclear which type of clinical specimens is most suitable for ZIKV detection. Investigations of small series of patients suggest that ZIKV is present in blood only a few days after infection. According to these studies, a generally low level of viral loads may further complicate ZIKV detection (11, 18). ZIKV detection in saliva may be more sensitive than detection in blood, but shedding in saliva and blood appeared to be equally short-lived (19). Urine, semen and saliva were reported to be positive for ZIKV RNA for 2 weeks and longer, and could thus be useful non-invasive materials for diagnosis and clinical studies (18, 20, 21). Here we determined viral load profiles in blood and urine, provide comparative laboratory data for published real-time RT-PCR tests, generate quantitative controls and project a high risk of false negative ZIKV test results. 5 Materials and Methods Clinical specimens Clinical specimens were obtained from routine diagnostics sent for investigation of ZIKV or DENV to the University of Bonn Medical Centre, Bonn, Germany, the Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany, the Academic Medical Centre, Amsterdam, the Netherlands and the Erasmus Medical Centre, Rotterdam, the Netherlands. Virus quantification and characterization DENV RNA quantification and flavivirus typing were done as described previously (22)(23). Quantitative controls were generated as described previously (24). The universal control RNA (ucRNA) was custom designed as a gBlocks fragment with a T7 promotor sequence (Integrated DNA Technologies, Leuven, Belgium) and in-vitro transcribed as described before (24). All individual IVT and the ucRNA allowed highly comparable quantification of ZIKV RNA with a mean 2-fold deviation of results (maximum deviation, 6-fold), suggesting the ability to use all of these controls to generate comparable results even upon usage of different real-time RT-PCR methods in different laboratories. For all other experiments, ZIKV RNA was generally quantified using reaction conditions exemplified in Supplementary Figure S1 . The ucRNA offers advantages to laboratories operating different real-time RT-PCR assays, but bears the same risk of laboratory contamination as full viral RNA. In contrast to full viral RNA, potential cases of laboratory contamination with the ucRNA can be proven by two highly sensitive real-time RT-PCR marker assay variants designed to specifically detect the ucRNA at lower limits of detection that were comparable to ZIKV-specific assays with 4.3 (95% confidence interval (CI), 2.9-10.9) and 3.3 (95% CI, 2.4-6.5) copies per reaction, respectively.