Journal of Clinical Virology 50 (2011) 4–7
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Journal of Clinical Virology
journal homepage: www.elsevier.com/locate/jcv
Cervicovaginal shedding of hepatitis C viral RNA is associated with the presence
of menstrual or other blood in cervicovaginal fluids
Chia C. Wanga,∗, Linda Cookb, Kenneth A. Tapiaa, Sarah Holtea,c, Meighan Krowsb, Arthur Bagabagb,
Anna Santosb, Lawrence Coreya,b,c, Keith R. Jeromeb,c
aDepartment of Medicine, University of Washington, Seattle, WA, United States
bDepartment of Laboratory Medicine, University of Washington, Seattle, WA, United States
cThe Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research Center, United States
a r t i c l ei n f o
Received 25 June 2010
Received in revised form
14 September 2010
Accepted 14 September 2010
Hepatitis C virus
a b s t r a c t
Background: The role of sexual activity in hepatitis C virus (HCV) transmission remains controversial.
Studies to date have not explored the relationship between HCV shedding in cervicovaginal fluids and
the presence of menstrual or other blood.
Objectives: Since cross-sectional studies may underestimate the prevalence of viral shedding, we per-
formed a 56-day longitudinal study of cervical HCV shedding.
Study design: Women self-collected cervicovaginal swabs for 56 consecutive days, while keeping a diary
of menses and genital symptoms. Swabs were tested for HCV RNA and cellular DNA by quantitative PCR,
and hemoglobin by spectrophotometry.
Results: Sixteen women contributed a total of 701 cervicovaginal swabs (mean collection period 48
days, range 18–56). Detection of HCV RNA was associated with detection of hemoglobin. Premenopausal
women were more likely than post-menopausal women to have HCV RNA detected in cervicovaginal flu-
ids. For premenopausal women, detection of HCV RNA was more likely during menstruation (OR=56.4)
or when hemoglobin was detected in cervicovaginal fluids, even if menstruation was not occurring
(OR=35.4). No woman post-hysterectomy had HCV RNA detected in cervicovaginal fluids on any day,
regardless of whether hemoglobin was detected.
Conclusions: Our findings are consistent with a low likelihood of sexual transmission of HCV. The results
suggest that shedding of HCV RNA in the female genital tract is associated with the presence of blood,
and requires the presence of a cervix. Clinicians should consider advising premenopausal women who
are concerned about transmitting infection that infectivity may increase during menstruation.
© 2010 Elsevier B.V. All rights reserved.
While injection drug use is recognized as the primary mode of
risk factors, including number of sex partners2and history of sexu-
ally transmitted diseases,3–5are associated with an increased risk
of HCV infection, yet studies of monogamous serodiscordant het-
erosexual couples have repeatedly demonstrated a lack of sexual
∗Corresponding author at: Virginia Mason Medical Center, 1100 Ninth Avenue,
Box C7N-ID, Seattle, WA 98101, United States. Tel.: +1 206 341 0846;
fax: +1 206 223 6814.
E-mail address: email@example.com (C.C. Wang).
There is some evidence that sexual transmission of HCV may be
of seven acute HCV infections in a sexual network of men who have
unprotected active and passive fisting were associated with HCV
increase the risk of HCV transmission.
Because of the lack of evidence of sexual transmission of HCV
in monogamous partners, the CDC has not recommended barrier
precautions for long-term monogamous couples.10For hetero-
sexual discordant sexual partnerships in which the woman is
HCV-infected, no specific recommendations regarding transmis-
sion risks during menstruation have been made. However, advice
regarding the safety of sex during menses is commonly sought by
couples concerned about transmission of HCV, and this potential
mode of transmission begs further study.
1386-6532/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
C.C. Wang et al. / Journal of Clinical Virology 50 (2011) 4–7
Several cross-sectional studies have reported detection rates
of HCV RNA in cervicovaginal fluids ranging from 011to 36%.12,13
Two studies specifically excluded cervical samples that were con-
taminated by hemoglobin assessed by spectrophotometry14or
by the presence of erythrocytes assessed microscopically.12The
remainder did not assess the presence of blood or hemoglobin in
connection with detection of HCV RNA.13–16Thus, studies to date
have not explored the relationship between HCV shedding in cer-
vicovaginal fluids and the presence of menstrual or other blood in
the female genital tract.
Longitudinal studies of HIV, HSV, HHV-8, and HCV have demon-
strated that virus shedding in body fluids is often intermittent;
as such, cross-sectional studies may substantially underestimate
the true prevalence of viral shedding.17–20We therefore chose
to implement a longitudinal study on cervical shedding of HCV,
since daily collection of cervicovaginal fluids allows evaluation of
the pattern of HCV shedding in relationship to menstrual flow for
each study participant. Here we present data on detection of HCV
RNA in cervicovaginal fluids in pre-menopausal, post-menopausal,
and post-hysterectomy women who collected daily cervicovaginal
swabs over 56 days.
3. Study design
This study was approved by the University of Washington Insti-
tutional Review Board. Female patients with chronic HCV were
levels at baseline. Women were instructed to insert two Dacron
swabs into the vagina every morning until they met resistance at
the back of the vagina, to rotate the swabs in a circle, and to wait at
least 1min before removing. Participants then placed the swabs
into dry cryovials, snapped off the plastic handles, capped and
placed the cryovials immediately into their home freezers. Women
collected swabs for 56 consecutive days. After day 56 the swabs
were transported on dry ice to the laboratory and stored at −80◦C
until testing. Subjects completed a daily diary indicating the occur-
rence of menses, genital tract symptoms, and lesions. All subjects
were tested for HIV and HSV-2 serostatus.
3.2. HCV RNA detection
To prepare cervical swabs for quantitative PCR, 200?l of phos-
10?l frozen for detection of hemoglobin. The remaining sample
was added to 300?l of Roche MagNApure lysis buffer containing
10mg/ml proteinase K and extracted using the Roche MagNAPure
reactions were performed on the purified nucleic acid: HCV RNA,
internal control plasmid (pAW109) added to the lysis buffer during
the extraction procedure, and betaglobin to quantitate the amount
of cellular DNA present in each sample.21
3.3. Validation of HCV RNA detection on swabs
of a high-level HCV-positive plasma sample were absorbed onto
swabs. The swabs were removed and extracted either immediately
or after being frozen overnight. Linear viral yields were obtained
and were indistinguishable between fresh and frozen specimens.
To confirm the ability of the assay to detect cell-associated virus,
HCV+ RNA replicon cells (Clone A, Apath, LLC, St. Louis, MO) were
used to create another serial dilution series. 200?l of the cell sus-
pension was absorbed onto swabs, and the extraction procedure
performed. Again, linear viral yields were observed indistinguish-
able from that of cell-free virus. The 95% detection limit for HCV
after extraction from swabs was <40IU/ml. Swabs with absorbed
trols for PCR runs over 3 months as part of this study. The swabs
ing the stability of frozen swab specimens. Post-study analysis of
subject data showed no statistically significant change in the like-
lihood of detecting HCV RNA on swabs during the 56 day study
period (OR 1.02, 95% confidence interval (0.99, 1.04)), demonstrat-
ing the validity of home storage of frozen specimens. To confirm
that hemoglobin or blood did not interfere with the PCR, 10?l
of HCV RNA-whole blood was added to the cell suspension dilu-
tions before absorption onto swabs. We observed no interference
compared to suspensions without the addition of whole blood.
3.4. Hemoglobin detection on cervical swabs
Ten microliters of the PBS used to resuspend the swab material
was analyzed with a BioTek PowerWaveX microplate spectropho-
tometer for the detection of Hemoglobin. The sample was diluted
with 190?l of PBS and then absorbance was read at 415nm (oxy-
hemoglobin) and 380nm (turbidity correction) using a method
modeled after plasma hemoglobin detection but excluding the
bilirubin measurement (450nm).22For each sample, a corrected
OD was calculated by the equation, corrected OD=[O.D. Sample
415nm−O.D. Blank 415nm]−[O.D. 380nm−O.D. Blank 380nm].
A 3-point hemoglobin standard curve with points at 60, 6, and
0.6mg/dL, constructed from a patient sample with hemoglobin of
14.5g/dl, was read simultaneously with the patient samples on a
Sysmex XT-2000i analyzer. Samples with hemoglobin >6.0mg/dl
were considered positive.
3.5. Other laboratory testing
Serum HCV RNA was measured using the Bayer Quantiplex HCV
RNA 2.0 Assay. Testing for hepatitis B surface antigen was per-
formed using the Abbott AuzymeTMassay. Serologic assays for HIV
and HCV were performed using the Genetic Systems HIV-1/HIV-2
Peptide EIA assay (Bio-Rad Laboratories) and the Abbott HCV EIA
by the UW Virology Laboratory at Seattle Children’s Hospital.
3.6. Statistical methods
Mixed effect methods24for logistic regression (with outcome
detectable HCV RNA) or linear regression (with outcome level of
HCV RNA) were used to account for repeated measures from the
4.1. Study population
Sixteen women contributed a total of 701 cervical swabs over
a mean collection period of 48 days (range 18–56 days), with 12
women collecting swabs on every day of the 56-day collection
period (Table 1). The median age was 31 (range 17–82 years),
C.C. Wang et al. / Journal of Clinical Virology 50 (2011) 4–7
Demographics, laboratory characteristics, and frequency of detection of HCV RNA in cervicovaginal fluids (N=16).
SubjectAge StatusBaseline serum, log10IU/mlGenotypeHSV2 HIV# swabs collected% swabs HCV+
and most participants were white (84%). One participant was HIV-
positive and seven women were HSV-2 seropositive. The median
serum HCV load was 6.4log10IU/ml (range 5.17–7.36log10IU/ml),
and 14 women had genotype 1 HCV infection. Seven women were
4.2. Detection of HCV RNA in cervicovaginal fluids
Thirty of 699 (4.3%) cervical swabs were positive for HCV
RNA (Fig. 1), with a median viral load of 4,693IU/ml (range
231–69,580IU/ml). 53 of the 699 swabs (7.6%) were positive for
Hb. All of the HCV-positive swabs were collected from 6 of the 16
women participating in the study; swabs from the remaining 10
women were uniformly negative. When cervicovaginal shedding
of HCV RNA was examined univariately in a logistic mixed effects
serum viral load, or HSV serostatus and cervicovaginal shedding of
HCV RNA. However, detection of Hb was associated with detection
of HCV RNA on cervicovaginal swabs, with a three times greater
likelihood of detection of HCV RNA on days when Hb was detected
(95% confidence interval 1.20–7.71).
When cervicovaginal HCV RNA was examined quantitatively
in a linear mixed effects model, the mean viral load of HCV
RNA detected when Hb was detected in cervicovaginal fluids
was 0.45log10IU/ml compared to 0.12log10IU/ml when Hb was
not detected (p<.001). Thus, there was a mean increase of
0.33log10IU/ml or 2.16IU/ml of HCV RNA detected in the sam-
ples with detectable Hb compared to those without (95% CI
women to have HCV RNA detected in cervicovaginal fluids on
at least 1 day (86% versus 16%, p=0.02). For premenopausal
women, detection of HCV RNA was more likely during menstru-
ation (OR=56.4, 95% CI (23.0, 138.3) or when hemoglobin was
detected in cervicovaginal fluids, even when menstruation was not
occurring (OR 35.4, 95% CI 12.2, 102.6). Interestingly, no woman
who had had a hysterectomy had HCV RNA detected in cervico-
vaginal fluids on any day, regardless of whether hemoglobin was
Overall, detection rates of HCV RNA on cervical swabs were low,
lected. Many of these women had participated in a previous study,
in which HCV RNA was detected in 72% of saliva samples.20Thus,
detection rates of HCV RNA in cervicovaginal fluids were markedly
lower than in saliva.
We report lower rates of HCV RNA detection than other pub-
lished studies. However, since most published studies did not
sitive methods to detect blood, it is difficult to compare our results
with published data. Regardless, our findings are consistent with
epidemiologic data supporting a low likelihood of sexual transmis-
sion of HCV.
For premenopausal women, detection of HCV RNA on cervi-
cal swabs was strongly associated with menstruation (OR=56.4,
95% CI 23.0, 138.3) and with detection of hemoglobin, even when
menstruation was not occurring (OR=35.4, 95% CI 12.2, 102.6). In
premenopausal women, HCV RNA was detected on 5 out of 318
days when hemoglobin was not detected, versus on 12 out of 31
Fig. 1. Detection of hemoglobin and HCV RNA on cervical swabs collected over 56 days. Red indicates days that hemoglobin was detected; yellow indicates days that HCV
RNA was detected; black indicates days that swabs were not collected.
C.C. Wang et al. / Journal of Clinical Virology 50 (2011) 4–7
days when hemoglobin was detected. In addition, the quantity of
virus detected on days with hemoglobin present was significantly
higher than on days when hemoglobin was absent. In one pre-
vious study, 15cc of menstrual blood was collected on the first
day of menses in 10 women with hepatitis C, and HCV RNA was
detected in all samples.25To our knowledge, this is the only other
published study examining the potential role of menstrual blood
on the presence of HCV RNA in the female genital tract. Taken
together, these two studies suggest that shedding of HCV RNA in
the female genital tract is more likely to occur in the presence of
HCV RNA was not detected on any day in any woman who had
had a hysterectomy, even though hemoglobin was detected on 16
of 136 (12%) cervical swabs collected from such women. Belec et
al. demonstrated that the presence of HCV RNA in cervicovaginal
secretions was restricted to cellular fractions containing cervical
cells as well as genital tract lymphocytes and monocytes.14Manavi
et al. localized the presence of HCV RNA to cervical lymphocytes,
but not to cervical epithelial cells or cervical granulocytes.13In
light of these past studies, our data suggest that the presence of a
cervix is necessary for shedding of HCV RNA in female genital tract
In conclusion, our data suggest that shedding of HCV RNA from
the genital tract is more frequent in premenopausal women than
in postmenopausal women or women who have had hysterec-
tomies. In premenopausal women, HCV RNA is associated with the
menopausal women, detection of HCV RNA in cervicovaginal fluids
seling chronically HCV-infected women who are concerned about
transmitting infection to their uninfected partners should consider
advising women about the possibility that infectivity may increase
during menstruation. Some women may choose to abstain from
intercourse or use barrier precautions during their menses.
Funding: Supported by grants from the National Institutes of
Health (K23 AI-51523 and U19 AI-31448) and internal funds.
Competing interests: None declared.
Ethical approval: Approved by the University of Washington
Institutional Review Board.
We thank Joanne Estergren for assistance with methods to
detect hemoglobin on cervical swabs, and Rachel Tompa for assis-
tance editing the manuscript.
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