The n e w e ng l a n d j o u r na l of
Original Article
From the National Center for Emerging
and Zoonotic Infectious Diseases (G.P.-B.,
J.M.-J., G.A.S., J.P.-P., F.A.M., S.H.W., L.E.A.,
M.L., T.M.S.) and the National Center for
HIV/AIDS, Viral Hepatitis, Sexually Trans-
mitted Diseases, and Tuberculosis Pre-
vention, Centers for Disease Control and
Prevention (K.D.), Atlanta; the Depart-
ment of Epidemiology and Biostatistics,
University at Albany School of Public
Health, SUNY, Albany, NY (E.S.R.); the
Oak Ridge Institute for Science and Edu-
cation, Oak Ridge, TN (L.K.); and Auxilio
Mutuo Hospital, San Juan (J.B.-P), and
Ponce Health Sciences University School
of Medicine–Saint Luke’s Episcopal Hos-
pital Consortium, Ponce (C.G.G., L.I.A.)
— both in Puerto Rico. Address reprint
requests to Dr. Paz-Bailey at the Centers
for Disease Control and Prevention, 1324
Calle Cañada, San Juan, PR 00920-3860,
or at ­gmb5@​­cdc​.­gov.
A preliminary version of this article was
published on February 14, 2017, at NEJM
.org.
N Engl J Med 2018;379:1234-43.
DOI: 10.1056/NEJMoa1613108
Copyright © 2018 Massachusetts Medical Society.
1234
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m e dic i n e
Persistence of Zika Virus in Body Fluids
— Final Report
Gabriela Paz‑Bailey, M.D., Ph.D., Eli S. Rosenberg, Ph.D., Kate Doyle, M.P.H.,
Jorge Munoz‑Jordan, Ph.D., Gilberto A. Santiago, Ph.D., Liore Klein, M.S.P.H.,
Janice Perez‑Padilla, M.P.H., Freddy A. Medina, Ph.D.,
Stephen H. Waterman, M.D., M.P.H., Laura E. Adams, D.V.M.,
Matthew J. Lozier, Ph.D., Jorge Bertrán‑Pasarell, M.D., Carlos Garcia Gubern, M.D.,
Luisa I. Alvarado, M.D., and Tyler M. Sharp, Ph.D.​​
A BS T R AC T
BACKGROUND
To estimate the frequency and duration of detectable Zika virus (ZIKV) RNA in human
body fluids, we prospectively assessed a cohort of recently infected participants in
Puerto Rico.
METHODS
We evaluated samples obtained from 295 participants (including 94 men who pro-
vided semen specimens) in whom ZIKV RNA was detected on reverse-transcriptase–
polymerase-chain-reaction (RT-PCR) assay in urine or blood at an enhanced arbo-
viral clinical surveillance site. We collected serum, urine, saliva, semen, and vaginal
secretions weekly for the first month and at 2, 4, and 6 months. All specimens were
tested by means of RT-PCR, and serum was tested with the use of anti–ZIKV IgM
enzyme-linked immunosorbent assay. Among the participants with ZIKV RNA in
any specimen at week 4, collection continued every 2 weeks thereafter until all
specimens tested negative. We used parametric Weibull regression models to esti-
mate the time until the loss of ZIKV RNA detection in each body fluid and reported
the findings in medians and 95th percentiles.
RESULTS
The medians and 95th percentiles for the time until the loss of ZIKV RNA detection
were 15 days (95% confidence interval [CI], 14 to 17) and 41 days (95% CI, 37 to 44),
respectively, in serum; 11 days (95% CI, 9 to 12) and 34 days (95% CI, 30 to 38) in
urine; and 42 days (95% CI, 35 to 50) and 120 days (95% CI, 100 to 139) in semen.
Less than 5% of participants had detectable ZIKV RNA in saliva or vaginal secretions.
CONCLUSIONS
The prolonged time until ZIKV RNA clearance in serum in this study may have
implications for the diagnosis and prevention of ZIKV infection. In 95% of the men
in this study, ZIKV RNA was cleared from semen after approximately 4 months.
(Funded by the Centers for Disease Control and Prevention.)
n engl j med 379;13 nejm.org  September 27, 2018
The New England Journal of Medicine
 Persistence of Zik a Virus in Body Fluids
A 
fter the discovery of Zika virus
(ZIKV) in Uganda in 1947, its transmis-
sion was limited until it was identified in
Brazil in 2015 and subsequently spread through-
out the Americas.1 ZIKV is now recognized as a
cause of congenital neurologic birth defects, no-
tably microcephaly,2 and has been associated with
potentially fatal complications.3,4
ZIKV infection can be diagnosed through de-
tection of ZIKV RNA in blood, urine, and other
body fluids by reverse-transcriptase–polymerase-
chain-reaction (RT-PCR) assay.5 However, the fre-
quency with which ZIKV RNA can be detected in
various body fluids and the length of time that
it remains detectable are not well understood.
Similarly, ZIKV infection can also be diagnosed by
the detection of anti–ZIKV IgM antibodies, al-
though the kinetics of IgM antibody production
have not been fully described.6
Although most ZIKV infections are transmitted
by infected mosquitoes, ZIKV transmission has
been documented as occurring through sexual
contact,7 blood transfusion,8 laboratory exposure,1
and both intrauterine and intrapartum transmis-
sion.9 ZIKV RNA has been detected in semen,10
urine,11 saliva,12 cerebrospinal fluid,13 vaginal or
cervical secretions,14,15 and other body fluids.16-19
Most transmissions through sexual contact have
been from men with symptomatic infection to
their female partners.20-22 However, sexual trans-
mission has also occurred from asymptomatic
men,23,24 through male-to-male25 and female-to-
male sex,26 and possibly through oral sex.10 Shed-
ding in the female genital tract appears to be of
short duration.15,27,28 In contrast, there are reports
of prolonged detection of ZIKV RNA in semen,
with the longest reported duration of detection
being up to 370 days after onset.29,30 Infectious
virus has been reported in semen up to 69 days
after onset.31,32 ZIKV infection has been associat-
ed with a decreased sperm count,33 but the overall
effect of infection on fertility is unclear.
An understanding of the dynamics of the early
stages of ZIKV infection can inform diagnostic
testing algorithms and prevention interventions,
since existing evidence is based on case reports,
small studies, and cross-sectional observations,
primarily involving returning travelers.34 To esti-
mate the presence and duration of the detection
of ZIKV RNA in body fluids and anti–ZIKV IgM
antibody among participants with acute ZIKV in-
fection, we established the ZIKV Persistence (ZiPer)
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cohort study in Puerto Rico, in which we prospec-
tively evaluated multiple concurrently obtained
specimens from participants. Here, we report the
final results of these analyses to inform recom-
mendations.
Me thods
Study Design and Oversight
ZiPer was a prospective cohort study involving
participants of all ages with ZIKV infection, as
diagnosed by means of RT-PCR, with a target en-
rollment of 350 participants. Beginning in May
2016, participants were identified through the
Sentinel Enhanced Dengue Surveillance System
(SEDSS), a prospective surveillance of acute febrile
illness among patients presenting to the emer-
gency department of a tertiary care hospital or an
outpatient clinic, both located in Ponce, Puerto
Rico.35 In April 2017, two more emergency depart-
ments were added in Guayama and San Juan,
Puerto Rico. Participation was offered to patients
who presented with fever (temperature, ≥38.0°C),
rash, conjunctivitis, or arthralgia. Among the par-
ticipants who provided written informed consent,
blood (all four study sites) and urine (the two sites
in Ponce only) specimens were tested for causative
agents of acute febrile illness, including ZIKV.
Participants with positive results for ZIKV in-
fection by RT-PCR (index participants) were sys-
tematically contacted by study staff and were of-
fered enrollment in ZiPer. Index participants were
defined as symptomatic participants enrolled at
emergency departments who had positive RT-PCR
results in any body fluid. The household members
of index participants were invited to participate
and provide specimens, and those who tested
positive on RT-PCR joined the prospective cohort
study. Full details regarding the study design are
provided in the protocol (available with the full
text of this article at NEJM.org), which was re-
viewed and approved by the institutional review
boards at the Centers for Disease Control and
Prevention (CDC) and Ponce Health Sciences Uni-
versity.
Procedures
All the participants completed an interviewer-
administered questionnaire, which included re-
porting the number of days that had elapsed since
the onset of ZIKV symptoms; household contacts
of the participants reported such data at the time
1235
 The n e w e ng l a n d j o u r na l of m e dic i n e

of enrollment. Participants were defined as being
symptomatic if they reported having had signs
or symptoms of ZIKV infection (fever, conjuncti-
vitis, rash, or arthralgia) during the 30 days be-
fore the interview. Serum, urine, saliva, semen,
and vaginal swabs (the last two in adults only)
were collected weekly for the first month and at
2, 4, and 6 months thereafter. Among the partici-
pants in whom ZIKV RNA was detected in any
specimen at week 4, biweekly collection continued
until all the specimens tested negative. All the par-
ticipants received a $50 reimbursement per visit.
Laboratory Testing
Specimens were refrigerated at 4°C upon collec-
tion and transported within 48 hours to the CDC
Dengue Branch laboratory in San Juan. Semen and
saliva specimens were frozen at −70°C upon arrival
at the laboratory, and other specimen types were
kept refrigerated at 4°C until tested. Hurricane
Maria caused catastrophic damage to Puerto Rico
when it landed on September 20, 2017. However,
the hurricane did not affect storage and processing
of specimens. Vaginal swabs and saliva (the latter
only when the specimen was not sufficient) were
diluted in viral transport medium. Specimens
(measuring 200 μl) were tested by means of the
Trioplex RT-PCR assay, as recommended by the
CDC for the detection of dengue, chikungunya,
and ZIKV RNA.36 In addition, we performed vali-
dation analyses for the use of the Trioplex RT-PCR
assay in semen37 (see the Supplementary Appendix,
available at NEJM.org). Specimens were consid-
ered to be positive if target amplification was de-
tected within 38 amplification cycles (cycle-thresh-
old values ≤38). Viral loads were expressed as log10
genome equivalents per milliliter of sample. The
RNA extraction and real-time RT-PCR process were
considered to be valid if the human RNase P reac-
tion was positive. Intermittent ZIKV RNA detection
was defined as the detection of viral RNA that was
followed by a lack of detection and then subse-
quent detection, regardless of the interval between
specimen collections. Serum was tested by means
of anti–ZIKV IgM antibody capture enzyme-linked
immunosorbent assay.38 ZIKV isolation was at-
tempted through culture in a subset of semen and
serum specimens.39
Statistical Analysis
We summarized the demographic and clinical Among the 1528 patients with symptomatic ZIKV
characteristics of the participants, along with de- infection confirmed by RT-PCR, 252 were enrolled
tails regarding the detection of ZIKV RNA in
fluids and IgM antibody according to the number
of days that had elapsed since the onset of ZIKV
symptoms. Analyses of urine specimens were re-
stricted to the 75% of participants recruited from
the two study sites in Ponce, since urine was not
collected at the first screening visit at the other
two sites. We used the kappa statistic to assess
the beyond-chance agreement in RNA detection
in paired samples of semen and serum and in
paired samples of semen and urine obtained from
male participants. Since all the participants had
positive results on testing of serum or urine at
enrollment, we could not independently assess the
serum–urine agreement. The time until the loss
of RNA detection in each fluid was defined as the
number of days between the onset of ZIKV symp-
toms and the first negative RT-PCR result. To es-
timate model-derived percentiles for the time until
virus clearance at the population level, we assumed
that all infected participants had ZIKV RNA in all
specimens at symptom onset. For the participants
who had intermittent shedding of ZIKV, we used
the first negative result after the final recorded test
result that was positive on RT-PCR.
We used the Kaplan–Meier method to estimate
survival functions for these outcomes, along with
the nonparametric maximum-likelihood Turnbull
estimator and parametric Weibull regression mod-
els. (Details about these models are provided in the
Supplementary Appendix.) The Turnbull method
and Weibull models accounted for interval censor-
ing (since the loss of detection of ZIKV RNA oc-
curred within an interval between visits instead of
being observed on an exact date). From the Weibull
models, we estimated survival functions and their
95% confidence intervals, as well as medians and
95th percentiles. In supplementary analyses (see
the Supplementary Appendix), we estimated mod-
els for the time until the loss of detection that
were restricted to the participants with any ZIKV
RNA in a given fluid and to index participants.
Model-derived medians were not estimated for
saliva and vaginal secretions because of the few
positive results. All statistical analyses were per-
formed with the use of SAS software, version 9.3.
R e sult s
Participant Characteristics

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 Persistence of Zik a Virus in Body Fluids

as index participants in the study. The percentage enrollment modestly influenced the time until the
of index participants who were adults (≥18 years loss of ZIKV RNA detection. When the analyses
of age) was higher among those who were enrolled were restricted to the 48% of participants who
in the study than among those who were not were enrolled within 4 days after symptom onset,
enrolled (84% vs. 73%), and more enrolled par- the median time until the loss of detection was
ticipants were male (48% vs. 40%). Among the 13 days (95% CI, 10 to 15). Among the 9 pregnant
369 household contacts of the index participants women, 4 had detectable RNA at 49 days after
who were screened, 43 (12%) tested positive for symptom onset. The maximum window of detec-
ZIKV RNA, for a total of 295 prospectively fol- tion among all participants was in a pregnant
lowed participants included in the analyses. participant for whom the last positive result was
Participants attended 1936 of 2065 visits (94%). at 83 days and the first negative result at 97 days
Four participants were lost to follow-up, 10 par- after symptom onset (Fig. S2A in the Supplemen-
ticipants withdrew, and 2 were discontinued by tary Appendix).
study staff owing to safety concerns.
The mean age of the participants was 36 years; ZIKV RNA in Urine
51% were female, including 9 who were pregnant ZIKV RNA was detected in at least one urine
(Table 1). Sixteen household contacts with positive specimen in 136 of 231 participants (59%) who
results were asymptomatic at enrollment. One provided urine specimens (Table 2). In 222 par-
index participant did not provide a valid date for ticipants with both urine and serum specimens,
onset of illness. Among the 280 participants with 26 (12%) had detectable RNA in urine but not in
signs or symptoms of ZIKV infection at enroll- serum, whereas 92 (41%) had RNA in serum but
ment, 88% were enrolled within 1 week after the not urine. The model-based median time until the
onset of illness. loss of detection was 11 days (95% CI, 9 to 12),
and the 95th percentile of time was 34 days
Antibody Response (95% CI, 30 to 38) (Fig. 1B).
Anti–ZIKV IgM antibody was detected in at least
one specimen obtained from 284 of 294 partici- ZIKV RNA in Saliva and Vaginal Secretions
pants (97%). Ten participants (3%) did not have Among the 291 participants who provided saliva
seroconversion. Two participants had a single visit, specimens and were tested, 14 (5%) had ZIKV RNA
and we therefore could not document seroconver- in at least one specimen (Table 2). The rate of
sion. Among the other 8 participants, the number positivity in these samples was lower than the rate
of follow-up visits ranged from 5 to 10 visits (rangein serum and urine at any number of days after
of days after symptom onset, 3 to 216). Two par- symptom onset (Table 3). Similarly, only 2 of 119
ticipants were RT-PCR–positive in urine only, 5 in women (2%) had ZIKV RNA in vaginal secretions
serum only, and 1 in urine and serum. (1 was an asymptomatic participant and 1 was
positive 3 days after symptom onset). All the
ZIKV RNA in Serum specimens were positive in the RNase P control
Among participants with a serum specimen avail- reaction.
able for RT-PCR, 251 of 284 (88%) had detectable
ZIKV RNA in at least one specimen (Table 2). Of ZIKV RNA in Semen
the 284 participants, 56 (20%) had detectable Of 117 eligible male participants, 94 (80%) pro-
ZIKV RNA more than once. Viral load results are vided at least one semen specimen. ZIKV RNA was
provided in Figure S7A in the Supplementary Ap- present in at least one specimen in 48 participants
pendix. The median time until the loss of RNA (51%). Chance-corrected agreement of RNA de-
detection was 15 days (95% confidence interval tection was low in paired samples of semen and
[CI], 14 to 17), and the 95th percentile of time was serum (κ = 0.11; 95% CI, 0.05 to 0.17) and speci-
41 days (95% CI, 37 to 44) on the basis of the mens of semen and urine (κ = 0.13; 95% CI, 0.07
Weibull model (Fig. 1A). Results that were ob- to 0.19). The model-derived median time until the
tained with the use of the Turnbull model were loss of RNA detection was 42 days (95% CI, 35 to
similar to those obtained with the Weibull model 50), and the 95th percentile of time was 120 days
(Fig. S1A in the Supplementary Appendix). The (95% CI, 100 to 139) (Fig. 1C). At 90 days, 11%
number of days after the onset of symptoms at had detectable RNA in semen. The maximum win-

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 The n e w e ng l a n d j o u r na l of m e dic i n e

Table 1. Characteristics of the Participants at Baseline.

dow of RNA detection was a last positive result
at 191 days, with the first negative result at 232
Participants days after symptom onset (Fig. S2C in the Sup-
Characteristic (N = 295)
plementary Appendix).
Age
Mean (range) — yr 36.3 (<1 to 83) Time until the Loss of Detectable ZIKV RNA
Age group — no./total no. (%)*
We also estimated the time until the loss of RNA
0–17 yr 50/294 (17.0)
detection among the participants with any ZIKV
18–64 yr 221/294 (75.2)
RNA in a given fluid. Among this subset, the
≥65 yr 23/295 (7.8)
model-derived estimated percentiles of time until
Female sex — no. (%) 151 (51.2)
the loss of RNA detection were longer because the
Pregnancy — no. (%) 9 (3.1)
analyses were limited to participants with viral
Presence of signs or symptoms of Zika virus
infection at enrollment — no. (%) shedding (Figs. S3, S4, and S5 in the Supplemen-
No† 15 (5.1) tary Appendix).
Yes 280 (94.9)
Days after symptom onset at enrollment —
Analyses Limited to Index Participants
no./total no. (%) Index participants were more likely to be female
0–2 days 44/280 (15.7) than were the 28 symptomatic household contacts
3–5 days 126/280 (45.0) (51% vs. 43%), to be at least 18 years of age (84%
6–7 days 75/280 (26.8) vs. 79%), and to have been recruited within 1 week
8–14 days 21/280 (7.5) after symptom onset (94% vs. 29%). When the
≥15 days 14/280 (5.0) model was limited to index participants, the esti-
Signs or symptoms at enrollment — no./total no. (%)‡ mated median time until the loss of detection of
Fever 219/278 (78.8) ZIKV RNA decreased by less than 1 day in serum,
Red eyes or eye pain 228/280 (81.4) urine, and semen (Fig. S6 in the Supplementary
Rash 265/278 (95.3) Appendix).
Pruritus 226/278 (81.3)
Photophobia 121/278 (43.5) Intermittent RNA Detection
Edema 172/277 (62.1) We observed intermittent ZIKV RNA detection in
Arthralgia 83/142 (58.5) serum obtained from 25 participants, in urine
Myalgia 205/259 (79.2) specimens obtained from 15 participants, and in
Headache 214/278 (77.0) semen specimens obtained from 12 participants
Abdominal pain 132/278 (47.5) (Fig. S8 in the Supplementary Appendix). The
Lymphadenopathy 89/278 (32.0) range of days between positive specimens was
Diarrhea 107/278 (38.5) 14 to 142 in serum, 14 to 204 in urine, and 14 to
Nausea 113/278 (40.6) 36 in semen.
Vomiting 34/278 (12.2)
Pelvic pain 40/273 (14.7) Isolation of ZIKV
Dysuria 50/278 (18.0) ZIKV isolation was attempted in specimens with
Other§ 231/279 (82.8) viral loads of at least 5.3 log10 genome equivalents
Laboratory findings per milliliter, including 78 semen and 36 serum
Median white-cell count (range) per mm3 5100 (2100 to 40,000) specimens. Virus isolation was successful in 10%
Median platelet count (range) per mm3 212,000 (80,000 to 373,000)
(8 of 78) of semen specimens, with days since
Median hematocrit (range) — % 42.0 (30.9 to 51.9)
onset ranging from 15 to 38 and viral loads rang-
* The age of one participant was not known. ing from 6.11 to 8.76 log10 genome equivalents
† This category includes one participant with an invalid date of symptom onset per milliliter. Virus isolation was successful in
and two participants who were asymptomatic at baseline but in whom signs 3% (2 of 36) of serum specimens. One successful
or symptoms developed within 7 days after specimen collection.
‡ The median duration of fever was 3 days (range, 1 to 22); red eyes or eye pain, 3 days isolation from serum was in a specimen from an
(range, 1 to 43); rash, 4 days (range, 1 to 25); and pruritus, 4 days (range, 1 to 46). asymptomatic participant, and the second was
§ Other signs or symptoms included cough (in 30.9% of the participants), yel- in a specimen obtained 3 days after onset; viral
low eyes or skin (3.6%), difficulty urinating (8.3%), blood in urine (4.7%),
painful ejaculation (4.3% of men), and penile discharge (1.7% of men). load was 7.75 log10 genome equivalents per mil-
liliter for both specimens.

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 Persistence of Zik a Virus in Body Fluids

Table 2. Detection of ZIKV RNA in Body Fluids and Anti–ZIKV IgM Antibody in Serum, According to Subgroup.*

Anti–ZIKV IgM
Subgroup ZIKV RNA Antibody

Vaginal
Serum Urine Saliva Secretions Semen Serum

number/total number (percent)

All participants 251/284 (88.4) 136/231 (58.9) 14/291 (4.8) 3/119 (2.5) 48/94 (51.1) 284/294 (96.6)
Age
0–17 yr 40/49 (81.6) 21/40 (52) 2/48 (4) 0/4 0/1 46/50 (92)
18–64 yr 196/213 (92.0) 100/172 (58.1) 12/220 (6) 3/101 (3.0) 44/87 (51) 216/221 (97.7)
≥65 yr 14/21 (67) 15/19 (79) 0/22 0/13 4/6 (67) 21/22 (96)
Sex
Male 118/137 (86.1) 69/118 (58.5) 9/144 (6.3) NA 48/94 (51) 139/143 (97)
Female 133/147 (90.5) 67/113 (59.3) 5/147 (3.4) 3/119 (2.5) NA 145/151 (96)
Pregnancy 9/9 (100) 1/8 (12) 0/9 0/9 NA 9/9 (100)

* Analyses of urine specimens were limited to the 75% of participants recruited from the Sentinel Enhanced Dengue Surveillance System
(SEDSS), since urine was not obtained at the screening visit at the other two sites. Saliva, vaginal secretions, and semen are not obtained
as part of SEDSS. Therefore, few specimens of these types were available within 7 days after the onset of symptoms. NA denotes not appli-
cable, and ZIKV Zika virus.

Discussion methods and in the population included in the

In this study we obtained data on how long ZIKV
RNA takes to clear in persons with acute ZIKV
infection and detectable ZIKV RNA at enrollment.
The recruitment of participants was based main-
ly on an ongoing surveillance platform that en-
abled nearly 90% of participants to enroll within
the first week after symptom onset, which pro-
vided increased resolution for ZIKV RNA detection
starting soon after onset. In our study, half the
participants had detectable viral RNA in urine for
at least 1 week after symptom onset, in serum for
2 weeks, and in semen for more than 1.5 months,
whereas 5% or less had detectable viral RNA in
urine for 5 weeks, in serum for 6 weeks, and in
semen for 4 months. Conversely, ZIKV RNA was
infrequently detected in saliva and vaginal secre-
tions.
The CDC recommends RT-PCR testing of se-
rum and urine samples obtained from symptom-
atic participants less than 14 days after symptom
onset.5 Our results contrast with the findings of
other studies,11,34 which showed more frequent
detection of ZIKV RNA in urine than in serum.
However, the cited studies had small sample sizes
that limit generalizability. The discrepant results
may also be explained by differences in testing
analyses. A previous study showed frequent ZIKV
RNA detection in saliva within 5 days after symp-
tom onset.12 We detected ZIKV RNA infrequently in
saliva; however, we did not have enough specimens
to determine RNA detection early after onset.
IgM antibody was detected in almost all ZIKV-
infected participants in this study. Given the
high prevalence of previous flavivirus infection
in Puerto Rico,40 it is important to document im-
mune responses in other populations not exten-
sively exposed to flaviviruses. The usefulness with
regard to the specificity of IgM testing for diag-
nosis of ZIKV in geographic areas with discrepant
exposure to flavivirus requires further study.
In our study, the observed duration of ZIKV
RNA in serum was longer than detection times
reported for dengue virus. More than 90% of the
patients who are infected with any of the four
dengue viruses clear RNA within 10 days after the
onset of symptoms.41 Studies involving asymp-
tomatic blood donors with the use of transcription-
mediated amplification (a technique that is more
sensitive than RT-PCR) showed that the median
time until RNA clearance for West Nile virus was
13 days (95% CI, 12 to 15), an interval that is
similar to what we observed for ZIKV.42 Since the
cross-reactivity of antibodies between flaviviruses

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 The n e w e ng l a n d j o u r na l of m e dic i n e

A ZIKV RNA in Serum Figure 1. Time until the Clearance of Zika Virus RNA
100 in Serum, Urine, and Semen.
90 Shown are models of the time until the loss of Zika
virus (ZIKV) RNA detection after the onset of symp-
80 toms in serum (Panel A), urine (Panel B), and semen
70 (Panel C), as estimated with the use of Weibull regres-
sion. To estimate model-derived percentiles for the
60
Positive (%)

time until virus clearance at the population level, we

15.3 (95% CI, 13.7–16.9) assumed that all infected participants had ZIKV RNA
50
in all specimens at symptom onset. Also shown are
40
medians and 95th percentiles of the time until the loss
30 of detection, the key values that were reported in this
study. Blue shading denotes 95% confidence intervals.
20
Data for 14 participants who were asymptomatic at
10 40.6 (95% CI, 36.9–44.3) the time of enrollment and 1 participant with an inval-
0
id onset date were excluded from the estimates of the
0 15 30 45 60 75 90 105 120 135 150 time until the loss of RNA detection, since the number
of days after the onset of symptoms could not be de-
Days after Onset of Symptoms
termined.
B ZIKV RNA in Urine
100
limits the use of serologic analysis, we recruited
90 participants who had detectable RNA at enroll-
80 ment, a factor that could have contributed to in-
70 creased times until RNA clearance.
We found that 11% of men had detectable RNA
60
Positive (%)

10.6 (95% CI, 9.2–12.0)

at 90 days and 5% by 4 months. However, we were
50
only able to isolate ZIKV in semen specimens with
40 high viral loads, and the maximum time of detec-
30 tion of infectious virus in semen was 38 days. The
20
longest time after symptom onset at which ZIKV
has been detected through culture is 69 days.31,32
10
34.1 (95% CI, 29.8–38.3) However, most studies have shown no evidence of
0
0 15 30 45 60 75 90 105 120 135 150
infectious virus more than 30 days after symptom
onset.10,29,33,43,44 Documented cases of sexual trans-
Days after Onset of Symptoms
mission also show that the most infectious pe-
C ZIKV RNA in Semen riod is 2 weeks after symptom onset45 and that the
100 longest period from symptom onset in the male
90 partner to sexual transmission in the female part-
ner has been 44 days.46 Together these findings
80
suggest that RNA is not a reliable indicator of
70 infectious virus. However, isolation by tissue cul-
60 ture may well be an insensitive method. For ex-
Positive (%)

50
42.0 (95% CI, 34.6–49.5) ample, historical studies have shown that intra-
40
thoracic injection of toxorhynchites mosquitoes
is more sensitive than cell culture for the detec-
30
tion of infectious dengue virus.47 New methods are
20 119.7 (95%CI, 101.1–139.3) needed to more sensitively and efficiently evaluate
10 specimens for the presence of infectious ZIKV.48
0 Preliminary results from this study have in-
0 15 30 45 60 75 90 105 120 135 150 formed previous CDC recommendations on the
Days after Onset of Symptoms prevention of ZIKV sexual transmission.7 The CDC
has recently reviewed its sexual transmission rec-

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 Persistence of Zik a Virus in Body Fluids

Table 3. Detection of ZIKV RNA in Body Fluids and Anti–ZIKV IgM Antibody in Serum, According to the Number of Days after Symptom
Onset.*

Positivity and Days Anti–ZIKV IgM

after Symptom Onset ZIKV RNA Antibody

Vaginal
Serum Urine† Saliva Secretions Semen Serum

number/total number (percent)

Participant analyses
Any interval after 241/268 (89.9) 129/218 (59.2) 11/276 (4.0) 2/114 (1.8) 45/88 (51.1) 271/278 (97.5)
symptom
onset
0–7 days 211/235 (89.8) 103/180 (57.2) 4/8 (50) 1/2 (50) 1/1 (100) 22/98 (22.4)
8–15 days 26/51 (51) 19/35 (54.3) 1/26 (3.9) 0/12 5/7 (71.4) 121/129 (93.8)
16–30 days 45/221 (20.4) 27/174 (15.5) 4/218 (1.8) 0/76 32/63 (50.8) 212/219 (96.8)
31–45 days 19/245 (7.8) 7/201 (3.5) 3/249 (1.2) 0/98 32/81 (39.5) 237/245 (96.7)
46–60 days 5/215 (2.3) 2/172 (1.2) 1/215 (0.5) 0/94 15/62 (24.2) 192/208 (92.3)
>60 days 3/259 (1.2) 1/212 (0.5) 0/268 1/112 (0.9) 15/82 (18.3) 195/263 (74.1)
Specimen analyses
Any interval after 327/2384 (13.7) 169/1894 (8.9) 15/2089 (0.7) 2/860 (0.2) 174/640 (27.2) 1590/2103 (75.6)
symptom
onset
0–7 days 212/236 (89.8) 104/181 (57.5) 5/9 (55.6) 1/2 (50) 1/2 (50) 22/99 (22.2)
8–15 days 27/54 (50) 20/37 (54.1) 1/27 (3.7) 0/12 5/7 (71.4) 125/134 (93.3)
16–30 days 55/348 (15.8) 33/272 (12.1) 4/335 (1.2) 0/117 48/94 (51.1) 331/342 (96.8)
31–45 days 22/464 (4.7) 9/390 (2.3) 4/460 (0.9) 0/187 51/141 (36.2) 427/444 (96.2)
46–60 days 6/267 (2.2) 2/214 (0.9) 1/260 (0.4) 0/125 15/75 (20) 237/255 (92.9)
>60 days 5/1015 (0.5) 1/800 (0.1) 0/998 1/417 (0.2) 54/321 (16.8) 448/829 (54.0)

* The number of participants and specimens that were evaluated at each interval after the onset of symptoms varies because participants
were enrolled as they presented for surveillance or tested positive as household contacts. Data for the 15 household contacts who were
­asymptomatic at the time of enrollment were excluded from this analysis, since there was no known date of symptom onset.
† Analyses of urine specimens were limited to the 75% of participants recruited from the SEDSS, since urine was not obtained at the screen-
ing visit in the other two sites.

ommendations and shortened the minimum pe-
riod that men with possible ZIKV exposure should
use condoms or abstain from sex from 6 months
to 3 months.49 This revision is based on the ad-
ditional evidence regarding isolation of infectious
virus and the timing of sexual transmission among
sexual partners discussed above. Similarly, the
CDC recommends that women who have been
infected or exposed to ZIKV wait at least 8 weeks
from symptom onset or last exposure before at-
tempting conception.49 In our study, 95% of the
participants no longer had detectable ZIKV RNA
in serum at 6 weeks. Our data suggest that the risk
of intrauterine transmission among ZIKV-infect-
ed women who are trying to conceive toward the
end of an 8-week period after symptom onset is
small.
Despite model-based estimates suggesting that
sexual transmission contributes only modestly
to epidemic propagation,50,51 sexual transmission
could complicate efforts to prevent the transmis-
sion of ZIKV. Furthermore, sexual transmission
may be more likely to result in infection of the
fetus than mosquito transmission.52,53
Our study has several limitations. By enrolling
only participants with positive results for ZIKV
RNA in urine or serum on RT-PCR assay at base-
line and excluding those who were IgM-positive

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 The n e w e ng l a n d j o u r na l of m e dic i n e

only, we may have biased our findings by re-
cruiting persons who had a longer duration of
ZIKV RNA in serum or urine. However, when our
analyses were limited to participants who had
enrolled within 2 days after symptom onset, our
duration estimates were similar to those in the
overall sample. We determined the limit of detec-
tion of ZIKV RNA in semen, but we were unable
to evaluate the sensitivity of the test for saliva and
vaginal secretions with a similar validation study.
However, all semen, saliva, and vaginal swabs
tested positive for the RNase P internal control
reaction, which suggests that the RNA extraction
and conditions of the assay are probably not rea-
sons for the failure to detect ZIKV RNA in saliva
and vaginal secretions. Nonetheless, without
knowing the limit of the detection of the Trioplex
RT-PCR assay in these specimen types, negative
results should be interpreted with caution. To
estimate model-derived percentiles for the time
until virus clearance at the population level, we
assumed that all infected participants had ZIKV
RNA in all specimens at symptom onset. This
assumption resulted in shorter median and 95th
percentile estimates than if we had limited our
analyses only to participants with detectable ZIKV
RNA. Data that were obtained from symptomatic
participants may not be generalizable to all per-
sons infected with ZIKV.
In conclusion, our study provides a longitudi-
nal assessment of multiple body fluids to describe
the persistence of ZIKV RNA among infected par-
ticipants. The results provide preliminary evidence
that ZIKV RNA is present in serum for a longer
period than expected for its most closely related
flavivirus, a finding that has implications for diag-
nostic recommendations and prevention of trans-
mission.
The views expressed in this article are those of the authors
and do not necessarily represent the official position of the Cen-
ters for Disease Control and Prevention.
Supported by the Centers for Disease Control and Prevention
(CDC) and by the Research Participation Program at the CDC
administered by the Oak Ridge Institute for Science and Educa-
tion through an interagency agreement between the Department
of Energy and the CDC. Staffing support was provided by the
Center for AIDS Research at Emory University (National Institutes
of Health grant P30AI050409).
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank the study participants for their time and support of
this project and thank Dania Rodriguez-Vargas, Brad Bigger-
staff, John T. Brooks, Laura Youngblood, Candimar Colon, Tyree
Staple, Olga Lorenzi, ZiPer staff members, and Nabal Bracero
and Fernando Rodriguez of the GENES clinic for their contribu-
tions to the study.

References
1. Petersen LR, Jamieson DJ, Honein MA.
Zika virus. N Engl J Med 2016;​375:​294-5.
2. Rasmussen SA, Jamieson DJ, Honein
MA, Petersen LR. Zika virus and birth de-
fects — reviewing the evidence for causal-
ity. N Engl J Med 2016;​374:​1981-7.
3. Cao-Lormeau VM, Blake A, Mons S, et
al. Guillain-Barré syndrome outbreak as-
sociated with Zika virus infection in French
Polynesia: a case-control study. Lancet
2016;​387:​1531-9.
4. Sharp TM, Muñoz-Jordán J, Perez-Pa-
dilla J, et al. Zika virus infection associ-
ated with severe thrombocytopenia. Clin
Infect Dis 2016;​63:​1198-201.
5. Centers for Disease Control and Pre-
vention. Guidance for US laboratories
testing for Zika virus infection. 2017
(https://www​.cdc​.gov/​zika/​laboratories/​
lab​-­g uidance​.html).
6. Munoz-Jordan JL. Diagnosis of Zika
virus infections: challenges and opportu-
nities. J Infect Dis 2017;​216:​Suppl10:​S951-
S956.
7. Brooks JT, Friedman A, Kachur RE,
LaFlam M, Peters PJ, Jamieson DJ. Update:
interim guidance for prevention of sexual
transmission of Zika virus — United
States, July 2016. MMWR Morb Mortal
Wkly Rep 2016;​65:​745-7.
8. Musso D, Nhan T, Robin E, et al. Po-
tential for Zika virus transmission through
blood transfusion demonstrated during an
outbreak in French Polynesia, November
2013 to February 2014. Euro Surveill 2014;​
19(14).
9. Perez S, Tato R, Cabrera JJ, et al. Con-
firmed case of Zika virus congenital in-
fection, Spain, March 2016. Euro Surveill
2016;​21(24).
10. D’Ortenzio E, Matheron S, Yazdanpa-
nah Y, et al. Evidence of sexual transmis-
sion of Zika virus. N Engl J Med 2016;​374:​
2195-8.
11. Gourinat AC, O’Connor O, Calvez E,
Goarant C, Dupont-Rouzeyrol M. Detec-
tion of Zika virus in urine. Emerg Infect
Dis 2015;​21:​84-6.
12. Musso D, Roche C, Nhan TX, Robin E,
Teissier A, Cao-Lormeau VM. Detection of
Zika virus in saliva. J Clin Virol 2015;​68:​
53-5.
13. Rozé B, Najioullah F, Signate A, et al.
Zika virus detection in cerebrospinal fluid
from two patients with encephalopathy,
Martinique, February 2016. Euro Surveill
2016;​21(16).
14. Nicastri E, Castilletti C, Balestra P,
Galgani S, Ippolito G. Zika virus infection
in the central nervous system and female
genital tract. Emerg Infect Dis 2016;​22:​
2228-30.
15. Prisant N, Bujan L, Benichou H, et al.
Zika virus in the female genital tract. Lan-
cet Infect Dis 2016;​16:​1000-1.
16. Calvet G, Aguiar RS, Melo ASO, et al.
Detection and sequencing of Zika virus
from amniotic fluid of fetuses with mi-
crocephaly in Brazil: a case study. Lancet
Infect Dis 2016;​16:​653-60.
17. Dupont-Rouzeyrol M, Biron A,
O’Connor O, Huguon E, Descloux E. In-
fectious Zika viral particles in breastmilk.
Lancet 2016;​387:​1051.
18. Fonseca K, Meatherall B, Zarra D, et
al. First case of Zika virus infection in a
returning Canadian traveler. Am J Trop
Med Hyg 2014;​91:​1035-8.
19. Furtado JM, Espósito DL, Klein TM,
Teixeira-Pinto T, da Fonseca BA. Uveitis
associated with Zika virus infection.
N Engl J Med 2016;​375:​394-6.
20. Foy BD, Kobylinski KC, Chilson Foy
JL, et al. Probable non-vector-borne trans-
mission of Zika virus, Colorado, USA.
Emerg Infect Dis 2011;​17:​880-2.
21. Hills SL, Russell K, Hennessey M, et
al. Transmission of Zika virus through
sexual contact with travelers to areas of
ongoing transmission — continental
United States, 2016. MMWR Morb Mortal
Wkly Rep 2016;​65:​215-6.
22. Venturi G, Zammarchi L, Fortuna C,

1242 n engl j med 379;13 nejm.org  September 27, 2018

The New England Journal of Medicine

Downloaded from nejm.org by GALO ACOSTA on October 22, 2018. For personal use only. No other uses without permission.
Copyright © 2018 Massachusetts Medical Society. All rights reserved.
 Persistence of Zik a Virus in Body Fluids

et al. An autochthonous case of Zika due
to possible sexual transmission, Florence,
Italy, 2014. Euro Surveill 2016;​21:​30148.
23. Fréour T, Mirallié S, Hubert B, et al.
Sexual transmission of Zika virus in an
entirely asymptomatic couple returning
from a Zika epidemic area, France, April
2016. Euro Surveill 2016;​21(23).
24. Brooks RB, Carlos MP, Myers RA, et
al. Likely sexual transmission of Zika vi-
rus from a man with no symptoms of in-
fection — Maryland, 2016. MMWR Morb
Mortal Wkly Rep 2016;​65:​915-6.
25. Deckard DT, Chung WM, Brooks JT,
et al. Male-to-male sexual transmission of
Zika virus — Texas, January 2016. MMWR
Morb Mortal Wkly Rep 2016;​65:​372-4.
26. Davidson A, Slavinski S, Komoto K,
Rakeman J, Weiss D. Suspected female-
to-male sexual transmission of Zika virus
— New York City, 2016. MMWR Morb
Mortal Wkly Rep 2016;​65:​716-7.
27. Iannetta M, Lalle E, Musso M, et al.
Persistent detection of dengue virus RNA
in vaginal secretion of a woman returning
from Sri Lanka to Italy, April 2017. Euro
Surveill 2017;​22(34).
28. Sánchez-Montalvá A, Pou D, Sulleiro
E, et al. Zika virus dynamics in body flu-
ids and risk of sexual transmission in a
non-endemic area. Trop Med Int Health
2018;​23:​92-100.
29. Barzon L, Percivalle E, Pacenti M, et
al. Virus and antibody dynamics in travel-
ers with acute Zika virus infection. Clin
Infect Dis 2018;​66:​1173-80.
30. Nicastri E, Castilletti C, Liuzzi G, Ian-
netta M, Capobianchi MR, Ippolito G.
Persistent detection of Zika virus RNA in
semen for six months after symptom on-
set in a traveller returning from Haiti to
Italy, February 2016. Euro Surveill 2016;​
21:​21.
31. Arsuaga M, Bujalance SG, Díaz-Mené-
ndez M, Vázquez A, Arribas JR. Probable
sexual transmission of Zika virus from a
vasectomised man. Lancet Infect Dis
2016;​16:​1107.
32. García-Bujalance S, Gutiérrez-Arroyo
A, De la Calle F, et al. Persistence and in-
fectivity of Zika virus in semen after re-
turning from endemic areas: report of 5
cases. J Clin Virol 2017;​96:​110-5.
33. Joguet G, Mansuy JM, Matusali G, et
al. Effect of acute Zika virus infection on
sperm and virus clearance in body fluids:
a prospective observational study. Lancet
Infect Dis 2017;​17:​1200-8.
34. Bingham AM, Cone M, Mock V, et al.
Comparison of test results for Zika virus
RNA in urine, serum, and saliva speci-
mens from persons with travel-associated
Zika virus disease — Florida, 2016. MMWR
Morb Mortal Wkly Rep 2016;​65:​475-8.
35. Tomashek KM, Lorenzi OD, Andújar-
Pérez DA, et al. Clinical and epidemio-
logic characteristics of dengue and other
etiologic agents among patients with
acute febrile illness, Puerto Rico, 2012–
2015 PLoS Negl Trop Dis 2017;​ 11(9)
e0005859.
36. Zika virus emergency use authoriza-
tion: emergency use authorizations: trioplex
real-time RT-PCR assay. Silver Spring, MD:​
Food and Drug Administration, 2016
(https://www​.fda​.gov/​downloads/​
MedicalDevices/​Safety/​
EmergencySituations/​UCM491592​.pdf).
37. Santiago GA, Vázquez J, Courtney S,
et al. Performance of the Trioplex real-time
RT-PCR assay for detection of Zika, dengue,
and chikungunya viruses. Nat Commun
2018;​9:​1391.
38. Zika virus emergency use authoriza-
tion: emergency use authorizations: Zika
MAC-ELISA. Silver Spring, MD:​Food and
Drug Administration, 2016 (https://www​
.fda​.gov/​downloads/​MedicalDevices/​Safety/​
EmergencySituations/​UCM488044​.pdf).
39. Medina FA, Torres G, Acevedo J, et al.
Duration of infectious Zika virus in se-
men and serum. J Infect Dis 2018 Jul 27
(Epub ahead of print).
40. Mohammed H, Tomashek KM, Stra-
mer SL, Hunsperger E. Prevalence of anti-
dengue immunoglobulin G antibodies
among American Red Cross blood donors
in Puerto Rico, 2006. Transfusion 2012;​
52:​1652-6.
41. Hunsperger EA, Muñoz-Jordán J, Bel-
tran M, et al. Performance of dengue di-
agnostic tests in a single-specimen diag-
nostic algorithm. J Infect Dis 2016;​214:​
836-44.
42. Busch MP, Kleinman SH, Tobler LH,
et al. Virus and antibody dynamics in
acute West Nile virus infection. J Infect
Dis 2008;​198:​984-93.
43. Mead PS, Duggal NK, Hook SA, et al.
Zika virus shedding in semen of symp-
tomatic infected men. N Engl J Med 2018;​
378:​1377-85.
44. Huits R, De Smet B, Ariën KK, Van
Esbroeck M, Bottieau E, Cnops L. Zika vi-
rus in semen: a prospective cohort study
of symptomatic travellers returning to
Belgium. Bull World Health Organ 2017;​
95:​802-9.
45. Counotte MJ, Kim CR, Wang J, et al.
Sexual transmission of Zika virus and
other flaviviruses: a living systematic re-
view. PLoS Med 2018;​15(7):​e1002611.
46. Turmel JM, Abgueguen P, Hubert B, et
al. Late sexual transmission of Zika virus
related to persistence in the semen. Lan-
cet 2016;​387:​2501.
47. Tesh RB. A method for the isolation
and identification of dengue viruses, us-
ing mosquito cell cultures. Am J Trop
Med Hyg 1979;​28:​1053-9.
48. Feldmann H. Virus in semen and the
risk of sexual transmission. N Engl J Med
2018;​378:​1440-1.
49. Polen KD, Gilboa SM, Hills S, et al.
Update: interim guidance for preconcep-
tion counseling and prevention of sexual
transmission of Zika virus for men with
possible Zika virus exposure — United
States, August 2018. MMWR Morb Mortal
Wkly Rep 2018;​67(31):​868-71.
50. Gao D, Lou Y, He D, et al. Prevention
and control of Zika as a mosquito-borne
and sexually transmitted disease: a math-
ematical modeling analysis. Sci Rep 2016;​
6:​28070.
51. Yakob L, Kucharski A, Hue S, Ed-
munds WJ. Low risk of a sexually-trans-
mitted Zika virus outbreak. Lancet Infect
Dis 2016;​16:​1100-2.
52. Rowland A, Washington CI, Sheffield
JS, Pardo-Villamizar CA, Segars JH. Zika
virus infection in semen: a call to action
and research. J Assist Reprod Genet 2016;​
33:​435-7.
53. Duggal NK, McDonald EM, Ritter JM,
Brault AC. Sexual transmission of Zika
virus enhances in utero transmission in a
mouse model. Sci Rep 2018;​8:​4510.
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