in early december 2013 , during the zikv outbreak , a 44-year - old man in tahiti had symptoms of zikv infection : asthenia , low grade fever ( temperature from 37.5c to 38c ) and arthralgia .
eight weeks later , he described a second episode of symptoms compatible with zikv infection : temperature from 37.5c to 38c , headache on days 13 , and wrist arthralgia on days 57 .
the patient did not seek treatment , thus biological samples were not collected during the first 2 periods of illness .
the patient fully recovered from the second episode , but 2 weeks later he noted signs of hematospermia and sought treatment . because the patient had experienced symptoms of zikv infection some weeks before , he was referred to our laboratory in the institut louis malard , papeete , tahiti for zikv infection diagnostic testing .
the medical questionnaire revealed no signs of urinary tract infection , prostatitis , urethritis , or cystitis , and the patient stated that he did not had any recent physical contact with persons who had acute zikv infection .
we extracted rna using the nuclisens easymag system ( biomrieux , marcy letoile , france ) from 200 l of blood and from 500 l of semen and urine ; both were eluted by 50 l of elution buffer .
we tested blood and semen rna extracts using real - time reverse transcription pcr ( rrt - pcr ) as described using 2 primers / probe amplification sets specific for zikv ( 3 ) .
the rrt - pcr results were positive for zikv in semen and negative in blood , and confirmed by sequencing of the genomic position 8581138 encompassing the prm / e protein coding regions of zikv . the generated sequence ( genbank accession no .
km014700 ) was identical to those previously reported at the beginning of the zikv outbreak ( 3 ) .
three days later , we collected a urine sample , then a second set of blood and semen samples .
semen and urine from this second collection were not found to contain traces of blood by both direct and macroscopic examinations .
rrt - pcr detected zikv rna in the semen and urine , but not in the blood sample .
we quantified zikv rna loads using an rna synthetic transcript standard that covers the region targeted by the 2 primers / probe sets .
rna loads were : 2.9  10 copies / ml and 1.1  10 copies / ml in the first and second semen samples , respectively , and 3.8  10 copies / ml in the urine sample .
we cultured semen and urine as described for dengue virus cultured from urine ( 6 ) .
briefly , 200 l of each sample diluted in 200 l of 1% fetal calf serum ( fcs ) minimum essential medium ( mem ) were inoculated onto vero cells and incubated for 1 h at 37c ; inoculum was then removed and replaced by 1 ml of culture medium .
we also inoculated a negative control ( 200 l of 1% fcs - mem ) and a positive control ( 5 l of a zikv - positive serum diluted in 200 l of 1% fcs - mem ) .
the presence of zikv in the culture fluids was detected by rrt - pcr as described .
replicative zikv particles were found in the 2 semen samples but none were detected in the urine sample .
( 7 ) , but perinatal transmission ( 8) and potential risk for transfusion - transmitted zikv infections has also been demonstrated ( 9 ) .
( 10 ) , who described a patient who was infected with zikv in southeastern senegal in 2008 . after returning to his home in colorado , united states , he experienced common symptoms of zikv infection and symptoms of prostatitis .
four days later , he observed signs of hematospermia , and on the same day , his wife had symptoms of zikv infection . because the wife of the patient had not traveled out of the united states during the previous year and had sexual intercourse with him 1 day after he returned home , transmission by semen was suggested .
zikv infection of the patient and his wife was confirmed by serologic testing , but the presence of zikv in the semen of the patient was not investigated .
infectious organisms , especially sexually transmitted microorganisms including viruses ( human papillomavirus or herpes simplex virus ) , are known to be etiologic agents of hematospermia ( 11 ) . to our knowledge , before the report of foy et al .
( 10 ) and this study , arbovirus infections in humans had not been reported to be associated with hematospermia , and no arboviruses had been isolated from human semen .
we detected a high zikv rna load and replicative zikv in semen samples , but zikv remained undetectable by rrt - pcr in the blood sample collected at the same time .
these results suggest that viral replication may have occurred in the genital tract , but we do not know when this replication started and how long it lasted . the fact that the patient had no common symptoms of zikv acute infection concomitantly to hematospermia suggests that the viremic phase occurred upstream , probably during the first or second episode of mild fever , headache , and arthralgia .
the detection of zikv in both urine and semen is consistent with the results obtained in a study of effects of japanese encephalitis virus , another flavivirus , on boars .
the virus was isolated from urine and semen of experimentally infected animals , and viremia developed in female boars that artificially inseminated with the infectious semen ( 12 ) .
flaviviruses have also been detected in urine of persons infected with west nile virus ( wnv ) .
wnv rna was detected in urine for a longer time and with higher rna load than in plasma ( 13 ) .
wnv antigens were detected in renal tubular epithelial cells , vascular endothelial cells , and macrophages of kidneys from infected hamsters ( 14 ) , suggesting that persistent shedding of wnv in urine was caused by viral replication in renal tissue . dengue virus ( denv )
rna and denv nonstructural protein 1 antigen were also detected in urine samples for a longer time than in blood , but infectious denv has not been isolated in culture .
concluded that the detection of denv by rrt - pcr was useful to confirm denv infections after the viremic phase ( 6 ) .
also , yellow fever virus rna was isolated from the urine of vaccinated persons ( 15 ) , and saint louis encephalitis viral antigens , but not infective virus , have been detected in urine samples from infected patients ( 10 ) .
furthermore , the observation that zikv rna was detectable in urine after viremia clearance in blood suggests that , as found for denv and wnv infections , urine samples can yield evidence ofzikv for late diagnosis , but more investigation is needed .
 in december 2013 , during a zika virus ( zikv ) outbreak in french polynesia , a patient in tahiti sought treatment for hematospermia , and zikv was isolated from his semen . 
 zikv transmission by sexual intercourse has been previously suspected . 
 this observation supports the possibility that zikv could be transmitted sexually . 
The Study
Conclusions