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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: de Laval F, Matheus S, Labrousse T, Enfissi A, Rousset D, Briolant S. Kinetics of Zika viral load in semen. N Engl J Med 2017;377: DOI: /NEJMc

2 SUPPLEMENTARY APPENDIX Title Kinetics of Zika Virus Load in Semen Table of content ADDITIONAL METHODS... 2 IRB APPROVAL AND STUDY DESIGN STATISTICAL ANALYSIS... 4 ADDITIONAL REFERENCES... 4 TABLE S

3 Methods RNA extraction from serum, urine and semen samples 560 μl of lysis buffer was added to 150 μl of sample. Extraction was processed according to the manufacturers instructions using the QIAamp Viral RNA kit (Qiagen, Hilden, Germany). Real time RT-PCR for detection of ZIKV, DENV and CHIKV RNA The amplification was performed in a LightCycler 2.0 Instrument (Roche Diagnostics GmbH, Switzerland). For the detection of ZIKV RNA in serum, urine and semen samples, the RealStar Zika Virus RT-PCR Kit 1.0 (Altona Diagnostics GmbH, Hamburg, Germany) was used as described by D Ortenzio et al., , without any modification. ZIKV RNA quantification was done by using as reference the European Virus Archive Zika ZIKV reference strain (SKU: 001N-0164). The viral load in each sample was estimated as log copy number per ml, with a quantified, diluted range of supernatants from ZIKV cultures. DENV and CHIKV RNA were detected using the methods described respectively by Callahan et al., and Panning et al IRB approval and Study design IRB approval has been given by the Comité de Protection des Personnes Sud- Méditerranée I corresponding to the following study Etude descriptive prospective de la maladie à virus Zika au sein de la communauté de défense des Forces Armées en Guyane and has been registered in february the 29 th 2016, under the number ID RCB: 2016-A Written informed consent was obtained from each patient as intended by the Comité de Protection des Personnes Sud-Méditerranée I. 2

4 In French Guiana, ZIKV outbreak occurred between January and October Between March and July 2016, we proposed to every patient with cutaneous rash, with or without fever, to perform ZIKV RT-PCR in serum and urine samples for ZIKV diagnosis. In case of ZIKV RT-PCR positivity, we proposed to ZIKV-infected patients to be enrolled in a longitudinal cohort survey. The inclusion criteria were: - To be military personnel or family member of military personnel. - To have ZIKV illness proven by ZIKV RNA detection on serum or urine samples. - To be at least 18 years old. - To be volunteer and give written consent. The exclusion criteria were: - To be pregnant. For ZIKV-infected male patients, we proposed specifically to give also sequentially semen samples. The primary study objective regarding sexual transmission aspects as designed was to evaluate the prevalence of men with Zika virus (ZIKV) detection in semen, the duration of ZIKV persistence in semen, and potential intermittent ZIKV excretion. The theoretical semen sample frame of men assessed was as follow: semen sampling at day five after onset of symptoms, at day 15 and then every fifteen days until negativity (with two consecutive negative samples to detect an eventual ZIKV intermittent excretion in semen). ZIKV infected patients gave semen samples according to the theoretical schedule but concretely in function of their availability and professional constraints. For initial diagnosis of ZIKV, urine and serum have been sampled at the first medical visit and at day 5 after onset of symptoms; as a part of a larger study, serum have also been sampled after onset of symptoms at days 3, 5, 7, 14, 21, 28, and then at month 2, 3, 6, 9 and 12. 3

5 Since the beginning of this study, 25 ZIKV symptomatic infected men have been enrolled, and only 12 have accepted to provide semen samples. Their median age was 39 years (range years), they had no comorbidity. Statistical analysis The correlation between each subject s highest ZIKV RNA viral load value in semen and serum was compared by a spearman correlation test, no correlation was observed between serum and semen ZIKV RNA viral loads (P = 0.09). In the eight patients with ZIKV RNA detection in semen, their initial viral load in semen (in copy number/ml) and their mean viral persistence in days (mean of the last day after onset of symptom with positive ZIKV RNA detection in semen and the first day after onset of symptom with negative ZIKV RNA detection in semen) was compared by a spearman correlation test, no correlation was observed between initial viral load in semen and persistence of ZIKV in semen (P = 0.24). Additional references 1. D'Ortenzio E, Matheron S, Yazdanpanah Y, de Lamballerie X, Hubert B, Piorkowski G et al. Evidence of sexual transmission of Zika virus. N Engl J Med. 2016; 374: Callahan JD, Wu SJ, Dion-Schultz A, Mangold BE, Peruski LF, Watts DM et al. Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus. J Clin Microbiol. 2001;39: Panning M, Grywna K, van Esbroeck M, Emmerich P, Drosten C. Chikungunya fever in travelers returning to Europe from the Indian Ocean region, Emerg Infect Dis. 2008;14:

6 Table S1: Kinetics of ZIKV loads in venous blood and semen samples from 12 patients, French Guiana, ZIKV load (log copy number/ml) Patients with no ZIKV RNA detection in semen Age yr Location of ZIKV infection Date of 1 45 Cayenne 03/18/2016 Days after Venous blood Semen NA NA 7 < 1.8 < < 1.8 < < 1.8 < < 1.8 < Rémire-Montjoly 03/20/ NA 4 < 1.8 < < 1.8 < Matoury 06/04/ Cayenne 06/03/2016 Patients with ZIKV RNA detection in semen Age yr Location of ZIKV infection Date of 5 34 Matoury 03/15/ Rémire-Montjoly 02/26/ Rémire-Montjoly 03/01/ Rémire-Montjoly 03/22/ Rémire-Montjoly 05/01/ Cayenne 06/03/ Cayenne 06/28/ Rémire-Montjoly 07/04/ NA 5 < 1.8 NA 10 < 1.8 NA 13 NA < < 1.8 NA 30 NA < < 1.8 (3.7)* NA 14 < 1.8 < < 1.8 < < 1.8 < 1.8 Days after NA: Not available, ZIKV: Zika virus, *in parenthesis: Zika virus load in urine sample. Venous blood Semen NA 3 < NA < NA < < 1.8 (3.6)* < < < 1.8 < NA < < < 1.8 < NA 3 < 1.8 NA 7 < < < 1.8 < NA NA 5 < NA < NA < NA < NA 6 < 1.8 NA 10 < 1.8 NA 17 < < < 1.8 < NA 3 < 1.8 NA 6 NA < 1.8 NA 15 NA < NA < NA < 1.8 NA 15 NA < NA NA < 1.8 Quantification was performed by real-time RT-PCR (RealStar Zika Virus RT-PCR Kit 1.0 CE; Altona Diagnostics GmbH, Hamburg, Germany) with a threshold of detection of 1.8 log copies per ml. 5

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