MAJOR ARTICLE
Detection of Hepatitis C Virus (HCV)
RNA in Normal Cervical Smears
of HCV-Seropositive Patients
Mahmood Manavi,1 Mehrdad Baghestanian,2 Thomas Watkins-Riedel,3 Walter Battistutti,4 Kerstin Pischinger,4
Christian Schatten,1 Eveline Witschko,1 Gernot Hudelist,1 Hanns Hofmann,3 and Klaus Czerwenka4
1
Department of Obstetrics and Gynecology, Division of Special Gynecology, 2Clinic of Internal Medicine II, Department of Angiology,
Department of Virology, and 4Department of Pathology, Division of Gynecopathology, University of Vienna, Austria
3
The presence of hepatitis C virus (HCV) in normal cervical smears (CS) obtained from 22 HCV-seropositive
and 50 HCV-seronegative patients was assessed by reverse-transcriptase–polymerase chain reaction (RT-PCR).
The presence of HCV in serum was established by use of enzyme-linked immunosorbent assay, Western blot
test, and RT-PCR. HCV was detected in 36.4% (n p 8 ) of CS cells recovered from 22 HCV-seropositive patients,
but not in CS samples obtained from 50 HCV-seronegative patients. Furthermore, cells from the CS of 2
seropositive/smear-positive patients and 1 seropositive/smear-negative patient were isolated; HCV RNA was
detectable in the cervical lymphocytes of the 2 smear-positive patients, but not in epithelial cells or granulocytes.
HCV RNA is detectable in the CS of some HCV-seropositive women. The clinical importance of these data
requires further study.
Hepatitis C virus (HCV), a positive-strand RNA virus
of ∼9.4 kb, is closely related to the flaviviruses and is
therefore different from hepatitis viruses A, B, or E [1].
It is postulated that HCV replication, like that of other
flaviviruses, requires the production of an antisense or
replicative-intermediate RNA (RNA-negative strand)
[2]. Thus, the presence of an RNA-negative strand
could be regarded as direct evidence of viral replication.
HCV infection is diagnosed by detection of HCVspecific antibodies in patient serum by means of serological assays [3] and by detection of HCV genomic
RNA (positive strand) in serum by means of molecular
assays, such as RT-PCR [4].
Although acute infection with HCV is usually
asymptomatic, ∼80% of patients eventually develop
Received 3 January 2002; revised 17 June 2002; electronically published 27
September 2002.
Reprints or correspondence: Dr. Mahmood Manavi, Dept. of Obstetrics and
Gynecology, Div. of Special Gynecology, University of Vienna, Waehringer Guertel
18-20, A-1090 Vienna, Austria ([email protected]).
Clinical Infectious Diseases 2002; 35:966–73
2002 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2002/3508-0008$15.00
966 • CID 2002:35 (15 October) • Manavi et al.
chronic infection [5]. Most carriers of chronic infection remain asymptomatic, but a significant number
of these patients go on to develop chronic liver disease
(i.e., cirrhosis, hepatocellular carcinoma, and liver
failure). Therefore, HCV-infected patients and their
sexual partners need to be aware of possible means
of transmission, particularly sexual transmission. Epidemiological studies have demonstrated that the most
efficient routes for the transmission of HCV are parenterally, by blood–blood product transfusion, by injection drug use, by occupational needle-stick injuries,
by hemodialysis, and through organ transplantation
[6, 7]. In ∼30%–40% of HCV cases, the route of transmission remains unknown [8].
Although infection with HCV through parenteral exposure is well documented, theories regarding sexual
transmission of HCV are controversial. Sexual transmission or other close human contact may play a role
in sporadic and community-acquired infections. The
high prevalence of HCV infection among prostitutes,
male homosexuals [9, 10], sexual partners of HCVinfected patients [11, 12], and patients who visit sexually transmitted disease clinics [13, 14] suggests that
Table 1.
Clinical data for patients whose normal cervical
smears were tested for hepatitis C virus (HCV).
Seropositive
patients
(n p 22)
Seronegative
patients
(n p 50)
40 17
32 9.2
II
II
Bilirubin, mg/dL
0.54 0.22
0.47 0.22
Lactate dehydrogenase, U/L
204 158.9
162 40.4
Alanine transferase, U/L
24 20.2
13 11.2
Aspartate transaminase, U/L
23 17
16 21.8
Characteristic
Age, years
Stage/grade of Papanicolaou smear
g-Glutamyl transferase, U/L
Alkaline phosphatase, U/L
No. of patients positive for HCV
NOTE.
29 34.5
18 36.7
129 102.8
114 65.6
8
0
Data are mean SD, unless otherwise indicated.
sexual transmission may occur [15, 16]. However, there are
studies that demonstrate no evidence of sexual transmission of
HCV [17, 18].
Although there is little doubt that HCV primarily replicates
in the liver [19, 20], theories regarding the presence of extrahepatic replication sites remain controversial. In support of
such a possibility are studies that have reported the relatively
common detection of HCV RNA–negative strands in mononuclear cells present in peripheral blood [21] or bone marrow
[22]. In addition, several publications report detection of HCV
RNA in semen [23, 24]. In contrast, in some other studies,
authors were unable to detect the HCV genome in seminal
fluid [25, 26]. The presence of HCV in seminal fluid would
provide further evidence for the biological plausibility of sexual
transmission of HCV. However, only one study has investigated
the presence of the HCV genome in normal cervical smears
(CS) [27]. To investigate possible transmission of HCV via
sexual contact, this study focused on the detection of HCV in
normal CS of HCV-seropositive and seronegative patients via
RT-PCR.
PATIENTS, MATERIALS, AND METHODS
Patient recruitment. From January 1995 through December
2000, premenopausal women seeking care at the Division of
Gynecology (University of Vienna, Austria) underwent serological screening for infection with hepatitis A, B, and C viruses
(HAV, HBV, HCV, respectively) and HIV, regardless of their
serum level of liver parameters. Twenty-two women who tested
HAV, HBV, and HIV seronegative but HCV seropositive by
means of ELISA were included in our study. In addition, 50
women were included who were HCV, HBV, HAV, and HIV
seronegative, as determined by ELISA. Routinely performed
cytological analysis of CS revealed neither signs of inflammation
nor cellular changes consistent with possible viral infections or
other infections. The clinical data for all patients are shown in
table 1.
CS. The materials for routine CS and for further analysis,
as described below, were obtained separately with swabs. Cytological analysis revealed representative amounts of epithelial
cells with an admixture of nonepithelial cells—for example,
RBCs (erythrocytes), WBCs (leukocytes), normal vaginal flora
(e.g., bacteria), and hemoglobin (protein background). Swabs
were transferred to tubes containing lysis solution. Equal parts
of the samples were stained for screening by Papanicolaou
smear. To exclude cross-contamination between paired samples,
preanalytical treatment of CS and detection of HCV RNA were
performed in strictly separate procedures.
Fluorescence in situ hybridization analysis (FISH). The
lymphocytes were stained with Giemsa stain to assess whether
⭓100 lymphocytic cells were present in CS. Decoloration was
performed with Carnoy solution (ratio of methanol to acetic
acid, 3:1). FISH was performed in accordance with the protocol
of Pinkel et al. [28]. The sequence of the 5-fluorescein labeled
oligonucleotide probe was 5-CCATAGTGGTCTGC GGAACCGGTGAGTACA-3 [29] (Boehringer Mannheim).
Staining of CS lymphocytes by immunofluorescence.
FISH-positive T lymphocytes were collected on slides and fixed.
Incubation with a monoclonal antibody against CD8 (anti–Leu2A; Becton-Dickinson) was performed. Cells were washed in
PBS. Isotype- and fluorochrome-matched negative controls
were included.
Isolation and purification of cells from CS. Epithelial cells,
lymphocytes, and granulocytes were isolated from CS samples
obtained from 3 patients (all patients were HCV seropositive:
2 of them were CS HCV positive and 1 was CS HCV negative).
The material was obtained with a cervical swab. The swab was
dipped into PBS (pH, 7.4). The probes were vortexed after 30
min. The obtained material was centrifuged and resuspended
in HPSS (1% HEPES; pH, 7.2 [Sigma] with 2% saccharose).
The cell pellet was placed in RBC buffer (RBC lysing buffer;
Sigma). Then the pellet was filtered through a 100-mm filter
(Schleicher and Schuell). The cells were washed off the filter
and added to a mixture of 0.5% of collagenase VII (Sigma)
and Dulbecco modified Eagle medium (Sigma). Cells were isolated with a 3-layer gradient (saccharose or Ficoll). Epithelial
cells (purity, 192%) were isolated from the upper bands of
1.046 g/mL gradient, granulocytes (purity, 93%) between the
1.077:1.119 and lymphocytes (purity, 197%) between the
plasma and 1.077 g/mL layers.
Detection and quantification of HCV RNA in peripheral
blood. Serum and plasma samples were prepared from whole
peripheral blood (WPB) specimens by means of centrifugation.
The sediment obtained after plasma preparation was also used
as the starting material and is hereafter referred to as “plasma
sediment.” All materials were stored at ⫺80C within !2 h.
HCV Genomic RNA in Cervical Smears • CID 2002:35 (15 October) • 967
RNA was isolated from plasma, WPB, or serum by use of the
TRI-ISO-RNA extraction kit (ViennaLab), as described elsewhere [30].
cDNA synthesis and amplification of HCV sequences.
Quantification of HCV RNA in total RNA was performed with
competitive RT-PCR by means of the HepaQuant-C protocol
(ViennaLab). HCV RNA isolated from blood was reverse-transcribed and amplified by means of primers specific for the HCV
5 noncoding region (NCR2: 5-ATACTGGAGGTGCCTTACGAGACCT-3). A reverse-transcriptase reaction was performed
for 45 min at 37C in a total volume of 50 mL containing 5
mL of test RNA, 10 pmol of specific primer recognizing HCV
plus strand RNA, 200 pmol of random hexamer primer, 100
U of Moloney murine leukemia virus–reverse transcriptase
(MMLV-RT), and 40 U of placental RNase inhibitor. Amplification of cDNA was performed for 40 cycles (94C for 30 s,
55C for 30 s, and 72C for 45 s) using a Perkin-Elmer Gene
Amp PCR System 9600. The total reaction volume of 50 mL
contained 10 pmol of biotinylated primer (NCR4: 5-biotinCACTCTCGAGCACCCTATCAGGCAGT-3; NCR5: 5-biotinGAGGAACTACTTCTTCACGCAGAA-3), 1 U of SuperTaq
(HT Biotechnology), and 5 mL of cDNA. Qualitative analysis
of HCV RNA in total RNA preparations was performed by use
of the HepaTest-C (ViennaLab).
HCV-specific amplification products were analyzed by size
fraction with 2.5% agarose gel and a microplate base hybridization system, as recommended by the manufacturer (HepaTest-C Detection Assay; ViennaLab). Results obtained by gelbased detection and the hybridization-based detection system
were compared with regard to sensitivity and specificity.
As an additional qualitative HCV PCR assay to confirm
HepaTest-C results, the Cobas Amplicor HCV-PCR test, version
2.0 (Roche Diagnostic Systems), was used in accordance with
the manufacturer’s instructions.
RT-PCR was monitored by use of CD44. The human CD44
gene was selected because it is moderately expressed in PBMCs,
and the amplification product resulting from the spliced mRNA
can be distinguished from others by its dimensions.
Determination of HCV genotypes. Genotyping of HCV
in patient samples was performed with the INNO-LiPA HCV
II (Innogenetics). Biotinylated amplification products generated by the HepaTest-C were hybridized to a panel of probes
immobilized on membrane strips. Sequence-specific binding to
the probes revealed the HCV-genotype of the samples after
detection of bound amplified material with avidin–alkaline
phosphatase conjugate and substrate.
Detection of Anti-HCV IgG in serum and plasma. AntiHCV IgG in serum and plasma was primarily determined by
means of Abbott HCV EIA 3rd Generation ELISA (Abbott
Diagnostics Division) following the manufacturer’s instruc-
968 • CID 2002:35 (15 October) • Manavi et al.
tions. In a second step, the ELISA results of all the women
included in our study were confirmed by use of a commercial
immunoblot assay kit (RecomBlot HCV IgG; Mikrogen).
RESULTS
HCV RNA in WPB, plasma, and CS samples. By use of 2
different sensitive and highly specific RT-PCR kits, we determined the HCV RNA genomic copies in WPB, serum, plasma,
and PBMCs (which contained all blood cell fractions). HCV
RNA could be detected in CS samples obtained from 8 (36.4%)
of 22 HCV-seropositive women. HCV genotypes in samples of
serum, WPB, and CS revealed no difference between corresponding samples obtained from each woman when duplicate
layout was performed. Table 2 shows the HCV RNA concentration in WPB and serum samples obtained from the 8 CSpositive and 14 (HCV-seropositive) CS-negative patients.
Table 2 shows the amount of nonepithelial cells (WBCs,
erythrocytes/hemoglobin, bacteria) and epithelial cells. Even in
patients with highly positive WPB samples, no HCV RNA could
be detected in CS (table 2). To examine the specificity and
sensitivity of the PCR methods used, CS from 50 HCV-seronegative women were examined. We did not detect HCV genomic RNA in any CS from this control group (table 1).
Figure 1 illustrates the HCV RNA genome copies determined
by RT-PCR from one typical seropositive, CS-positive patient.
Amplification of CD44 was performed for each patient as an
internal control. Distilled water served as a negative control for
RT-PCR.
Detection of HCV RNA in cellular subfractions of CS.
HCV RNA was further determined in cellular subfractions of
CS (cervical epithelial cells, lymphocytes, granulocytes) from 3
patients (2 were HCV seropositive and CS positive [patients 7
and 8], and 1 was HCV seropositive and CS negative [patient
12]). As shown in figure 2, the HCV RNA genome copies were
only found in cervical lymphocytes. Such copies were not found
in either cervical epithelial cells or in cervical granulocytes recovered from patients who were both HCV seropositive and
CS positive. The results of RT-PCR were confirmed by FISH
staining. Lymphocytes obtained by oil density isolation from
CS of the 2 seropositive and CS-positive patients and the 1
seropositive and CS-negative patient were further processed by
use of the FISH assay. Again, the expression of HCV RNA was
observed in the nuclei of lymphocytes from both of the CSpositive patients examined. By use of the fluorescence staining
technique, we could detect Leu-2A (CD8), a specific marker
for T cells, on 70% of these lymphocytes (data not shown).
Transcripts of HCV genome-specific RNA could not be detected
in any cellular subfraction of CS obtained from CS-negative
patients by RT-PCR (figure 2C) or FISH staining (figure 3).
Table 2.
Detection of hepatitis C virus (HCV) RNA in serum, whole peripheral blood, and cervical smear samples.
d
Serum/WPB/CS results, by test
Patient
a
b
Amount of n-epc/epc, % score
c
Amplicor HCV
1
2⫹/4⫹/2⫹
1.2 ⫻ 106/1 ⫻ 106/pos
3a/3a/3a
⫹1
⫹2
⫹1
⫹1
2
3⫹/4⫹/3⫹
2.7 ⫻ 106/3.3 ⫻ 106/pos
1b/1b/1b
⫹1
⫹1
⫹1
⫹2
3
2⫹/3⫹/1⫹
4.2 ⫻ 105/2.4 ⫻ 105/pos
1b/1b/1b
⫹2
⫹1
⫹1
⫹2
4
4⫹/4⫹/3⫹
3.2 ⫻ 106/2.7 ⫻ 106/pos
1b/1b/1b
⫹1
⫹1
⫹1
⫹1
5
3⫹/3⫹/2⫹
6.5 ⫻ 10 /6 ⫻ 10 /pos
1a/1a/1a
⫹1
⫹1
⫹1
⫹1
6
4⫹/4⫹/1⫹
1.1 ⫻ 106/1.3 ⫻ 106/pos
3a/3a/3a
⫹1
⫹1
⫹1
⫹1
7
3⫹/3⫹/2⫹
1.1 ⫻ 106/1.3 ⫻ 106/pos
3a/3a/3a
8
1⫹/1⫹/2⫹
1.5 ⫻ 103/3 ⫻ 103/pos
5
5
HCV genotyping
RBC
WBC
Microorganisms
Hemoglobin
(background)
Internal RT-PCR
⫹1
⫹1
⫹1
⫹1
Untypeable
⫹1
⫹1
⫹1
⫹1
9
2⫹/2⫹/neg
5.6 ⫻ 10 /3.3 ⫻ 10 /neg
1b/1b/neg
⫹1
⫹1
⫹1
⫹1
10
3⫹/4⫹/neg
6.3 ⫻ 105/1.1 ⫻ 106/neg
3a/3a/neg
⫹1
⫹1
⫹1
⫹1
11
3⫹/4⫹/neg
8.5 ⫻ 105/6 ⫻ 105/neg
3a/3a/neg
⫹1
⫹2
⫹1
⫹2
12
3⫹/3⫹/neg
2.5 ⫻ 105/1.5 ⫻ 105/neg
4h/4h/neg
⫹1
⫹2
⫹1
⫹2
13
2⫹/2⫹/neg
1.7 ⫻ 104/1.2 ⫻ 104/neg
4h/4h/neg
⫹2
⫹2
⫹1
⫹2
14
2⫹/2⫹/neg
3.1 ⫻ 10 /2.6 ⫻ 10 /neg
1a/1a/neg
⫹1
⫹1
⫹1
⫹1
15
3⫹/2⫹/neg
1.8 ⫻ 105/7.2 ⫻ 104/neg
3a/3a/neg
⫹1
⫹2
⫹1
⫹1
16
2⫹/3⫹/neg
5.3 ⫻ 104/1.4 ⫻ 105/neg
1b/1b/neg
⫹2
⫹1
⫹1
⫹2
17
3⫹/3⫹/neg
2.2 ⫻ 105/1.9 ⫻ 105/neg
3a/3a/neg
⫹1
⫹2
⫹1
⫹1
18
2⫹/2⫹/neg
3.8 ⫻ 10 /1.6 ⫻ 10 /neg
1a/1a/neg
⫹1
⫹1
⫹1
⫹1
19
1⫹/2⫹/neg
6.1 ⫻ 103/1.9 ⫻ 104/neg
3a/3a/neg
⫹1
⫹1
⫹1
⫹2
20
3⫹/2⫹/neg
2.3 ⫻ 105/7.2 ⫻ 104/neg
1a/1a/neg
⫹2
⫹1
⫹1
⫹2
21
2⫹/2⫹/neg
2.9 ⫻ 104/4.7 ⫻ 104/neg
1b/1b/neg
⫹1
⫹2
⫹1
⫹1
2⫹/1⫹/neg
1.7 ⫻ 10 /7.8 ⫻ 10 /neg
1a/1a/neg
⫹1
⫹2
⫹1
⫹1
22
NOTE.
5
4
4
4
5
4
4
3
CS, cervical smear; epc: epithelial cells; neg, negative; n-epc: nonepithelial cells; WPB, whole peripheral blood; pos, positive.
a
Semiquantitative estimation of HCV RNA titers of serum and WPB samples via densitometric gel analyses. Estimated ranges for WPB and serum are provided
according to a synthetic HCV RNA standard of 1.2 ⫻ 104 copies/mL and serial 10-fold dilutions of RNA extracts: neg !1.0 ⫻ 103 ; 1⫹ p 1.1 ⫻ 103 ⫺ 1.0 ⫻ 104 ; 2⫹
p 1.1 ⫻ 104 ⫺ 1.0 ⫻ 105; 3⫹ p 1.1 ⫻ 105 ⫺ 1.0 ⫻ 106; 4⫹ 11.0 ⫻ 106.
b
Results of quantitative estimation of HCV genomic copies in serum and WPB samples by use of the TRI-ISO RNA Extraction Kit (ViennaLab).
c
Assessed by Inno-LiPA HCV II (Innogenetics). Geno(sub)typing 1a,b; 2a,b,c; 3a,b; 4; 5; 6; nomenclature according to Simmonds et al. [31].
d
Score: ⫹1 p 0%–10%; ⫹2 p 10%–20%.
DISCUSSION
In this study, we examined the presence of HCV genomic RNA
in nonpathological CS from 22 HCV-seropositive and 50 HCVseronegative women by means of RT-PCR. Unfortunately, for
30%–50% of all HCV-positive patients, the route of transmission remains uncertain. The extent to which sexual transmission may account for HCV infection without evidence of parenteral exposure is controversial and still under discussion.
Several authors have denied that sexual transmission occurs
[17, 18], whereas others have presented data suggesting that
HCV can be transmitted sexually [15, 16]. Wyld et al. [18]
hypothesized that, if replication of HCV is confined to hepatocytes, it may be the absence of suitable target cells in the
genital tract that prevents infection with HCV through any
route other than the parenteral route. To address this hypothesis, we isolated and thoroughly purified epithelial cells, lymphocytes, and granulocytes. We managed to detect HCV ge-
nomic RNA in cervical lymphocytes, but not in granulocytes
or cervical epithelial cells recovered from CS. These data correlate with recent studies showing that subpopulations of
PBMCs are variably infected with HCV.
Zehender et al. [32] and, more recently, Crovatto et al. [33]
demonstrated that lymphocytes are more likely to be infected
with HCV than are granulocytes. In addition, B lymphocytes
were more frequently infected with HCV than were T lymphocytes. Furthermore, the replicative form of HCV RNA was
detected in B lymphocytes but not in T lymphocytes. The
reason B and T lymphocytes are not equally sensitive to HCV
remains unclear. Large populations of T lymphocytes are present in the cervix under normal conditions [34]. Cytotoxic
suppressor T cells are the major T cell subtype present in the
cervix. These lymphocytes can be found in both the epithelium and stroma of the exocervix and endocervix, but they
are predominantly present in the subepithelial stroma. Vari-
HCV Genomic RNA in Cervical Smears • CID 2002:35 (15 October) • 969
Figure 1. Hepatitis C virus (HCV) genomic RNA detection for a typical seropositive and cervical smear (CS)–positive patient. Samples of whole
peripheral blood (WPB), serum, plasma, and CS were obtained from one patient as described in Patients, Materials, and Methods. RNA was isolated
and analyzed by means of RT-PCR. HCV genomic RNA was detected in WPB (lane 1), serum (lane 2), plasma (lane 3), EDTA plasma sediments (lane
4), EDTA WPB (lane 6), and CS total (lane 9). Amplification of CD44 in EDTA-plasma sediment/blood cells (lane 5) and EDTA WPB (lane 7) was
performed as an internal control. Distilled water (lane 8) served as a negative control. Size markers (lane 10) indicate the number of base pairs (bp).
able numbers of T helper cells are also present in the exocervical stroma [35].
In our study, we did not determine the subgroup of HCV
RNA–positive cervical lymphocytes, but, by use of an immunofluorescence staining technique, we showed that 70% of cervical lymphocytes express surface-bound CD8. This might be
indirect evidence that the HCV-affected cervical lymphocytes
are T cells. Further experiments are necessary to confirm the
discrepancy observed in our study.
Several studies have investigated the presence of HCV in
saliva and seminal fluid, but, to our knowledge, this is the first
report providing evidence for the possible infection of cervical
lymphocytes with HCV. Numerous authors have reported that,
for detection of HCV RNA, RT-PCR of WPB samples was
significantly more sensitive than RT-PCR of plasma samples
[21, 34]. As a result, these authors recommended the determination of HCV content via PCR technique in the serum as
well as in the blood cells of each patient. In our study, 2 different
RT-PCR kits from 2 independent laboratories were used for
the detection of HCV RNA. For all patients in whom antibody
Figure 2. Detection of hepatitis C virus (HCV) genomic RNA in purified and enriched cervical lymphocytes. Cervical smear (CS) samples were
obtained from 1 HCV-seropositive and CS-negative patient and from 2 HCV-seropositive and CS-positive patients. Cervical lymphocytes obtained from
2 HCV-seropositive and CS-positive patients expressed HCV genomic RNA (A and B, lane 2), whereas the cervical lymphocytes from the HCV-seropositive
and CS-negative patient did not express any transcripts of HCV genome-specific RNA (C, lane 2). HCV genome-specific transcripts were not detected
in either granulocytes or in cervical epithelial cells isolated from CS of both HCV-seropositive and CS-positive patients (A and B, lanes 4 and 5) and
CS-negative patients (C, lanes 4 and 5). CD44 served as an internal RT-PCR–positive control for lymphocytes and granulocytes (lanes 1 and 3).
970 • CID 2002:35 (15 October) • Manavi et al.
Figure 3. Detection of hepatitis C virus (HCV) genomic RNA in cervical smear (CS) lymphocytes by means of fluorescence in situ hybridization
analysis (FISH). Cervical lymphocytes were sorted and enriched as described Patients, Materials, and Methods. FISH staining was performed by means
of an HCV-specific primer. HCV RNA was expressed and detectable in the nuclei of CS lymphocytes from a CS-positive patient (A; original magnification,
⫻200). HCV RNA was not detectable in lymphocytes from a CS-negative patient (B; original magnification, ⫻200).
against HCV was observed in serum samples by Western blot
test, we were able to find HCV RNA not only in WPB specimens
but also in plasma specimens. These data confirm the high
sensitivity and specificity of the methods used in our laboratory.
By use of these highly sensitive methods, we detected HCV
RNA in only 8 of 22 patients. As to the cause of this, HCV is
found in the cervical lymphocytes of approximately one-third
of seropositive patients. The difference between the extremely high proportion of HCV-positive lymphocytes in blood
(87%–94% positive B lymphocytes) and the low proportion in
cervical lavage may be due to differences in the location of the
lymphocytes (blood vs. cervical stromal lymphocytes) with reference to expression of their membrane-bound receptors. These
variable patterns of receptor expression might arise as a result
of local cytokines and growth factors. To date, there are no
studies that have addressed this question. Apart from the lowdensity lipoprotein receptor [36], which binds the envelope
protein E1 of HCV, and CD81 [37], which binds the major
envelope protein E2, no other receptors are known to be associated with the internalization of HCV into the cell. It is also
conceivable that additional, as yet unidentified, cellular proteins
are involved in virus binding and entry into various cells. Thus,
separate reservoirs of virus may exist, and HCV may use different receptors to access these different cell types. Therefore,
clarification of the superficial markers on cervical lymphocytes
would be of great interest.
In our study, none of the cervical epithelial cells isolated
from the CS (3 were from HCV-seropositive patients, 2 of
whom were CS positive and 1 of whom was CS negative) contained HCV RNA. We cannot say with any confidence whether
HCV Genomic RNA in Cervical Smears • CID 2002:35 (15 October) • 971
the cervical epithelial cells should be categorized as HCV target
cells. The findings of published articles are inconsistent with
regard the infection of epithelial cells by HCV. Taliani et al.
[38] found no evidence that HCV had infected salivary gland
epithelial cells in patients with HCV viremia and chronic liver
disease. More recently, however, by use of FISH and immunohistochemistry, Arrieta et al. [39] managed to detect HCV
RNA of both positive and negative polarity, as well as HCV
core antigen, in the epithelial cells of salivary gland biopsy
specimens obtained from HCV-positive patients.
We were unable to answer 2 questions of interest. First, we
wanted to know whether CS from patients with a higher concentration of HCV in WPB and/or plasma are more likely to
be CS positive than CS from patients with a lower virus content
in WPB and/or plasma. This finding would be in line with
statistical analyses that have revealed a direct relationship between the virus load of HCV and its infectiousness. Second,
we wanted to know whether certain HCV genome types (1a,
1b, and 3a) preferentially target cervical lymphocytes. These
questions remain to be answered. We have demonstrated that
HCV genomic RNA can be detected in 36% of CS from HCVseropositive women; HCV genomic RNA was present in cervical
lymphocytes, but neither cervical epithelial cells nor granulocytes were found to be targets of HCV infection.
Acknowledgments
We thank Monika Glitzner and Sabine Schwindlegger for
their invaluable technical assistance.
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