Hepatitis C Virus Infection in Spouses of Patients with
Type C Chronic Liver Disease
Annals of Internal Medicine, 1 May 1994; 120:
Yoshihiro Akahane, MD; Mineo Kojima, MD; Yoshiki Sugai, MD;
Minoru Sakamoto, MD; Yoshiki Miyazaki, MD; Takeshi Tanaka, BS;
Fumio Tsuda, PhD; Shunji Mishiro, MD; Hiroaki Okamoto, MD;
Yuzo Miyakawa, MD; and Makoto Mayumi, MD
Objective: Survey for markers of hepatitis C virus (Hepatitis C Virus)
infection in spouses of patients with Hepatitis C Virus-related chronic
Design: Cross-sectional clinical, serologic, and
molecular biological study of spouses of patients with Hepatitis C Virus
viremia and chronic liver disease.
Setting: University and city hospitals.
Participants: Spouses (52 men and 102 women; mean
age, 56 ± 11 years) of 154 patients with Hepatitis C Virus viremia (102 men
and 52 women; mean age, 58 ± 10 years), of whom 66 had
chronic hepatitis, 49 had liver cirrhosis, and 39 had primary
Methods: Tests for Hepatitis C Virus-associated antibodies were
done using a second-generation enzyme immunoassay and
immunoassays with synthetic oligopeptides deduced from the Hepatitis C Virus
core gene. Hepatitis C virus RNA was detected by polymerase
chain reaction with primers deduced from the 5'-noncoding
region and Hepatitis C Virus genotypes by reaction with type-specific
primers deduced from the Hepatitis C Virus core gene.
Results: Hepatitis C virus-associated antibodies
were detected in 42 (27%) spouses, of whom 25 were also
positive for Hepatitis C Virus RNA. Of 112 (73%) spouses without detectable
antibodies, 2 had chronic liver disease. The development of
markers of Hepatitis C Virus infection in spouses increased with the
duration of marriage, ranging from 1 to 60 years (30 ± 11
Conclusions: Spouses of patients with Hepatitis C Virus viremia
and chronic liver disease have an increased risk for acquiring
Hepatitis C Virus, which is proportional to the duration of marriage. They
should be followed routinely for markers of Hepatitis C Virus infection and
Ann Intern Med. 1994;120:748-752. Annals of
Internal Medicine is published twice monthly and
copyrighted © 1994 by the American College of Physicians.
Yamanashi Medical College, Yamanashi-Ken, Kojima Clinic,
Gifu-Ken; Iwaki Kyoritsu General Hospital,
Fukushima-Ken; Japanese Red Cross Saitama Blood Center,
Saitama- Ken; the Viral Hepatitis Research Foundation of
Japan, Tokyo; Institute of Immunology, Tokyo; Jichi
Medical School, Tochigi-Ken; and Mita Institute, Tokyo,
Japan. For current author addresses,
The discovery by Choo and colleagues
of hepatitis C virus (Hepatitis C Virus), the cause of most cases of
blood-borne non-A, non-B hepatitis worldwide, allowed more accurate diagnosis and prevention of Hepatitis C Virus
infection. Detection of antibody to Hepatitis C Virus (anti-Hepatitis C Virus) by enzyme
immunoassays and determination of Hepatitis C Virus RNA by polymerase chain
reaction (PCR) have proved useful in diagnosing Hepatitis C Virus-related
acute and chronic liver disease and in preventing
post-transfusion Hepatitis C Virus infection.
Hepatitis C virus is transmitted parenterally, typically by
transfusion, illicit intravenous drugs, and accidental needle
However, a defined parenteral exposure accounts for only one
half of the reported cases of acute hepatitis C .
Unlike hepatitis B virus infection, vertical transmission of
Hepatitis C Virus infection from mother to infant is rare. The route of Hepatitis C Virus transmission is unknown in most
patients with hepatitis C and in most symptom-free Hepatitis C Virus
carriers identified among blood donors by routine screening
for anti-Hepatitis C Virus.
Sexual transmission of Hepatitis C Virus has been investigated in
homosexual men , persons attending clinics for sexually transmitted
spouses or sexual partners of patients with acute or chronic
hepatitis C ,
and persons with hemophilia who are infected with Hepatitis C Virus .
These studies indicate that Hepatitis C Virus is transmitted sexually only
infrequently. Previous studies of sexual transmission of Hepatitis C Virus
usually have been done in settings in which sexual contact was
transient or in spouses married only for a limited time to
patients with hepatitis. The situation can differ greatly,
however, for spouses who have been married to patients with
Hepatitis C Virus- related chronic liver disease for many decades. To
determine if long-term spouses are at increased risk for Hepatitis C Virus
infection, we measured Hepatitis C Virus-associated antibodies and Hepatitis C Virus RNA
and determined Hepatitis C Virus genotypes in spouses of 154 patients with
Hepatitis C Virus-related chronic liver disease, including 84 persons
previously reported .
Spouses of consecutive patients at Yamanashi Medical
College, Kojima Clinic, and Iwaki Kyoritsu General Hospital
from October to December 1991 were studied. Most were referred
from satellite medical facilities and screened for Hepatitis C Virus-associated
antibodies. Patients with antibodies were tested for Hepatitis C Virus RNA.
One hundred fifty-four patients were positive for both
antibodies and Hepatitis C Virus RNA, including 66 patients with chronic
hepatitis, 49 with liver cirrhosis, and 39 with primary
hepatocellular carcinoma, of whom 102 were men (age, 58 ± 11
years) and 52 women (age, 57 ± 8 years). It was not known how
long the patients had had clinical hepatitis or were infected
with Hepatitis C Virus.
Their spouses, 102 women (age, 54 ± 12 years) and 52 men
(age, 60 ± 9 years), were tested for Hepatitis C Virus-associated
antibodies and Hepatitis C Virus RNA, and Hepatitis C Virus genotypes were determined.
They were asked to complete a questionnaire addressing the
duration of present marriage, history of transfusion,
occurrence of premarital non-A, non-B hepatitis, experiences
of illicit intravenous drugs, previous marriages, and
extramarital sexual relationships.
We also studied nine spouses who were excluded from the
cohort because they had risk factors other than marriage to
patients infected with Hepatitis C Virus, such as a history of transfusion
or premarital non-A, non-B hepatitis.
Antihepatitis C virus was screened using a
second-generation enzyme immunoassay (EIA-II, Ortho Diagnostic
Systems; Tokyo, Japan) with absorbance at 492 nm (A492)
values greater than 0.635 considered reactive. Antibody to a
synthetic 36-mer peptide representing amino acids 39-74 of the
product of the Hepatitis C Virus core gene (anti-CP9) and antibody to a
synthetic 19-mer peptide representing amino acids 5-23
(anti-CP10) were determined by EIA by methods described
previously and A492 values greater than 0.300 were
Hepatitis C Virus RNA
The detection of Hepatitis C Virus RNA by PCR was done using a slight
modification of the method described previously ,
with primers deduced from the 5'-noncoding region of the Hepatitis C Virus
genome. Briefly, nucleic acids were extracted from 100 mL of
serum, reverse-transcribed to cDNA using primer #299 (AC
CCAACACTACTCGGCTAG, antisense, nucleotides [nt] 250- 269) and
Moloney murine leukemia virus reverse transcriptase
(Superscript, GIBCO-BRL; Gaithersburg, Maryland), and
amplified by a two-stage PCR with AmpliTaq DNA polymerase
(Perkin-Elmer Cetus; Norwalk, Connecticut). The first PCR was
done with primer pair #32 (CTGTGAGGAACTACT GTCTT, sense, nt
45-64)/#299 for 35 cycles and the second PCR with #33 (TTCACGCAGAAAGCGTCTAG
nt 63-82)/#48 (GTTGATCCAAGAAAGGACCC, nt 188-207) for 25
cycles; nucleotides were numbered from the putative 5`-end of
the Hepatitis C Virus genome .
Each cycle included denaturation at 94 °C for 45 seconds,
primer annealing at 55 °C for 45 seconds, and primer
extension at 72 °C for 90 seconds. The Hepatitis C Virus RNA assay was at
least as sensitive as or 103-fold more sensitive
than that for detection of Hepatitis C Virus in chimpanzee transmission
experiments (Unpublished observations).
Hepatitis C Virus Genotypes
Hepatitis C virus RNA samples in spouses and those in
corresponding patients were classified in terms of genotypes
I, II, III, IV, and V .
A part of the Hepatitis C Virus core gene spanning nt 480-751 (272 base
pairs [bp]) was amplified on Hepatitis C Virus cDNA with universal primers,
#256 (CGCGCGACTAG GAAGACTTC nt 480-499) and #256V (CGCGCGACGCGTA
AAACTTC, nt 480-499)/#186 (ATGTACCCCATGAGGT CGGC, nt 732-751).
A portion of the product was amplified by PCR with two
universal sense primers, #104 (AGGAAGACT TCCGAGCGGTC nt
489-508) and #104V (CGTAAAACTTC TGAACGGTC, nt 489-508), and
the mixture of five antisense primers deduced from sequences
of the Hepatitis C Virus core gene, #296 (GGATAGGCTGACGTCTACCT, nt 518-537),
#133 (GAGC CATCCTGCCCACCCCA, nt 613-632), #134 (CCAAGAGG
GACGGGAACCTC, nt 643-662), #135 (ACCCTCG TTTCCG TACAGAG, nt
592-611), and #339 (GCTGAGCCCAGGAC CGGTCT, nt 557-576), which
were specific for genotypes I, II, III, IV, and V,
respectively. The five genotypes were distinguished from one
another by distinct sizes of PCR products: 49 bp for genotype
I; 144 bp for II; 174 bp for III; 123 bp for IV; and 88 bp for
A typing method was proposed by Chan and colleagues
and by Simmonds and coworkers
that classifies Hepatitis C Virus isolates into three major types-1, 2, and
3-based on the phylogenetic relatedness, with each type
divided into distinct subtypes. Subtypes 1a and 1b in this
classification correspond to I and II, respectively; 2a and 2b
to III and IV; and 3a and 3b to V and VI. In another
classification by Houghton and colleagues
and by Cha and coworkers ,
group I corresponds to genotype I (1a), group II to genotype
II (1b), and group III to genotypes III (2a) and IV (2b);
their group IV encompasses genotype V (3a) and group V
includes a herd of variants reported only from South Africa so
Serologic Testing for the
Hepatitis B surface antigen was determined by passive
hemagglutination with commercial assay kits (Mycell; Institute
of Immunology, Co., Ltd., Tokyo, Japan). Antibody to human
immunodeficiency virus type 1 was determined by passive
agglutination of gelatin microparticles coated with viral
antigens (SERODIA · HIV; Fuji Rebio, Tokyo, Japan).
Frequencies between groups were compared using the chi-
square test and the Fisher exact test. Exact confidence
intervals were obtained by the standard method .
Index patients with Hepatitis C Virus-related chronic liver disease
included 66 with chronic hepatitis, 49 with cirrhosis, and 39
with hepatocellular carcinoma. They all tested positive for
Hepatitis C Virus RNA as determined by PCR and for anti-Hepatitis C Virus by EIA-II. None
had hepatitis B surface antigen or antibody to human
immunodeficiency virus type 1.
The patients' 154 spouses were tested for markers of Hepatitis C Virus
infection. The spouses included 52 husbands and 102 wives.
None had been previously married, and all denied intravenous
drug abuse or extramarital sexual contacts. None had received
a transfusion or had a history of premarital non-A, non-B
Hepatitis C Virus Markers
in Spouses of Patients
Anti-Hepatitis C Virus as detected by EIA-II, anti-CP9, or anti-CP10 was
detected in 42 (27%) of 154 spouses, of whom 25 (16%) tested
positive for Hepatitis C Virus RNA. Of the remaining 112 spouses without
detectable Hepatitis C Virus-associated antibodies, 2 (2%) were positive for
Hepatitis C Virus RNA. No appreciable differences were found in the diseases
of the index patients married to spouses with Hepatitis C Virus markers and
to those without these markers. Hepatitis C virus markers were
detected in 17 (33%) of 52 husbands and in 27 (26%) of 102
wives of index patients.
Prevalence of Hepatitis C Virus antibodies and RNA increased in parallel
with the duration of marriage (Figure 1). Hepatitis C virus
markers were not detected in any of seven spouses who had been
married to patients for fewer than 10 years. An increase
therefore occurred in Hepatitis C Virus- associated antibodies and Hepatitis C Virus RNA
detected in spouses proportional to the duration of marriage.
Markers for Hepatitis C Virus were detected in three (60%) of five spouses
married longer than 50 years. Of 87 spouses married longer
than 30 years, Hepatitis C Virus RNA was detected in 21 (24%) compared with
only 6 (9%) of 67 married less than 30 years (P <
0.05). Hepatitis C virus antibodies were also detected more
frequently in the former group (28 of 87 [32%] compared with
14 of 67 [21%]). Logistic regression showed an increase in the
odds of anti-Hepatitis C Virus positivity of 50% per decade of marriage
(odds ratio, 1.5; 95% CI, 1.05 to 2.2; P = 0.01), and
the odds of Hepatitis C Virus RNA positivity increased by 90% per decade
(odds ratio, 1.9; 95% CI, 1.2 to 2.9; P = 0.004).
Of the 27 spouses with Hepatitis C Virus RNA, 24 (89%) were infected with
Hepatitis C Virus of genotypes identical to those of index patients . The remaining three spouses had Hepatitis C Virus of genotypes
different from those of patients. Thus, Hepatitis C Virus RNA of the
genotypes identical to those in the index patients was
detected in 24 (16%) of 154 spouses, including 14 (14%) of 102
wives and 10 (19%) of 52 husbands.
compares prevalences of Hepatitis C Virus antibodies and Hepatitis C Virus RNA in
spouses of index patients classified by Hepatitis C Virus genotypes. No
statistically significant differences were found in the
prevalence of Hepatitis C Virus markers in spouses of patients infected with
Hepatitis C Virus of genotype II, III, or IV, or with mixed genotypes (II
Liver Diseases in Spouses
Infected with Hepatitis C Virus
Of 24 spouses infected with Hepatitis C Virus of genotypes identical to
patients, 8 had previously noted liver disease. Liver biopsy
samples showed cirrhosis in one and chronic hepatitis in
seven. Two additional spouses had elevated transaminase levels
and were both found to have chronic hepatitis after liver
biopsy. Thus, 10 spouses with apparent liver disease tested
positive for Hepatitis C Virus-associated antibodies. The remaining 14 did
not have elevated alanine aminotransferase levels, but Hepatitis C Virus-associated
antibodies were detected in 12 (86%) of them. No appreciable
differences were found between the 10 spouses with apparent
liver disease and the 14 without liver disease in age (63 ± 8
compared with 56 ± 10 years) or duration of marriage (39 ± 8
compared with 32 ± 10 years).
Hepatitis C Virus Infection
in Spouses with Other Risk Factors
Nine spouses with Hepatitis C Virus antibodies were excluded from the
cohort because they had received transfusions or had a history
of non-A, non-B hepatitis before the marriage. Four were
positive for Hepatitis C Virus RNA. Hepatitis C virus genotypes were
identical to those of patients in only one. The rate of
identical Hepatitis C Virus genotypes in these four patients (1 of 4 [25%])
was lower than that (24 of 27 [89%]) in spouses without risk
factors other than marriage to patients (P < 0.04).
Hepatitis C virus-associated antibodies were detected in 42
(27%) and Hepatitis C Virus RNA in 27 (18%) of 154 spouses married to
patients with viremia and Hepatitis C Virus-related chronic liver disease
for periods of 1 to 60 years. The prevalence of Hepatitis C Virus antibodies
among them was much higher than 1.5% in the general population
and probably in age- and sex-matched subpopulations. In 24
(89%) of the 27 spouses with viremia, genotypes of Hepatitis C Virus were
identical to those of the index patients: Nineteen were
infected with Hepatitis C Virus of genotype II, four with genotype III, and
one with mixed genotypes (II and III). Although discordance of
Hepatitis C Virus genotypes may exclude the infection from suspected
sources, concordance may not necessarily identify the route of
infection. Restriction enzyme patterns and nucleotide
sequences of Hepatitis C Virus cDNA from patients and spouses with viremia
would have to be compared to establish transmission between
them. Such technical reservations notwithstanding, the 24
spouses in this study probably acquired Hepatitis C Virus infection from
their partners with Hepatitis C Virus- related chronic liver disease. This
view is supported by concordance of Hepatitis C Virus genotypes in only one
(25%) of four spouses with viremia who had risk factors other
than the marriage.
The risk for Hepatitis C Virus infection increased with duration of
marriage, ranging from 1 to 60 years (mean, 30 years). None of
the seven spouses married to patients for fewer than 10 years
had Hepatitis C Virus antibodies or Hepatitis C Virus RNA. The prevalence of Hepatitis C Virus markers
gradually increased with the duration of marriage. Three (60%)
of five spouses married for longer than 50 years had both
anti-Hepatitis C Virus and Hepatitis C Virus RNA. Taken along with the lack of other risk
factors for Hepatitis C Virus infection in the studied spouses, such as
transfusion, history of hepatitis, intravenous drug abuse,
extramarital sexual contact, or previous marriages, the
observed correlation between development of Hepatitis C Virus markers and
the duration of marriage over decades favors possible
transmission of Hepatitis C Virus infection from patients to spouses.
The correlation between the duration of marriage and the
development of Hepatitis C Virus infection in spouses presupposes that index
patients were already infected at the time of marriage.
However, because it is not known how long patients had
clinically apparent disease or were infected with Hepatitis C Virus, the
actual exposure time cannot be determined. Prospective studies
are needed to establish the correlation of Hepatitis C Virus infection and
the duration of sexual exposure but would require decades. A
common source of infection for both patients and spouses is
another possibility but would be rare considering the low rate
of infectivity of Hepatitis C Virus for community-acquired or household
infection, except between spouses. Sharing needles for illicit
intravenous drugs would be the only plausible explanation for
a common source infection, but none of the spouses or patients
studied were drug abusers.
Our results support those of previous studies in some ways
and contradict them in others. It has been claimed that sexual
transmission of Hepatitis C Virus is infrequent based on screening of Hepatitis C Virus-associated
antibodies in homosexual men and patients seen in sexually
transmitted disease clinics
Hepatitis C virus is clearly less transmissible than hepatitis
B virus or human immunodeficiency virus seen more frequently
in such patients with sex-associated risk factors. In our
study, Hepatitis C Virus infection was not found in any of the seven spouses
married for fewer than 10 years to patients with Hepatitis C Virus-related
chronic liver disease. Such a low rate of transmission to
spouses is remarkable, considering that Hepatitis C Virus RNA levels in
patients with chronic liver disease probably would be much
higher than those in symptom-free carriers having transient
extramarital sexual relations. Substantial rates of
transmission have not been observed after transient sexual
contacts to inconsistent partners with unspecified Hepatitis C Virus
or among spouses married to patients with acute hepatitis C
for 1 year or less.
Low estimates of risk have been based on studies documenting
the lack or paucity of Hepatitis C Virus- associated antibodies in spouses
or sexual partners of patients with chronic hepatitis C
in which the risk is not correlated with the duration of
marriage. Later studies have shown increased risk in family
members of patients with chronic hepatitis C in which spouses
have the highest risk , in agreement with our results. The term "sexual
contact" encompasses a wide range of relationships. It
includes extramarital affairs with changing partners for
limited periods, as well as lifetime commitment to only one
partner over many years. Low rates of sexual transmission of
Hepatitis C Virus observed in some situations may not be extrapolated to
others. Previous data therefore may not be applicable to
spouses married to patients with Hepatitis C Virus-related chronic liver
disease for many decades. Our results corroborate substantial
heterosexual transmission of Hepatitis C Virus recently proposed by others .
It is interesting that 10 (42%) of the 24 spouses with Hepatitis C Virus
infection, probably acquired after marriage, had overt
clinical disease. Eight had been attending liver clinics for
some time before the study. The remaining two were identified
by screening for Hepatitis C Virus markers and elevated transaminase levels
in the course of the study, and both had chronic hepatitis
diagnosed by liver biopsies. Because many Hepatitis C Virus-infected persons
may be asymptomatic and have normal aminotransferase levels,
and because liver biopsies were not done on the remaining 14
asymptomatic spouses infected with Hepatitis C Virus, we may have
underestimated the extent of liver disease in these persons.
Spouses of patients with Hepatitis C Virus viremia and chronic liver
disease have an increased risk for developing Hepatitis C Virus infection,
and the risk increases over time. Spouses married to persons
with demonstrable anti-Hepatitis C Virus, whether symptom-free carriers or
patients with documented liver disease, should be screened for
Hepatitis C Virus markers at regular intervals, perhaps twice a year, to
receive prompt care if and when they become infected.
Table 2. Hepatitis C Virus Markers in Spouses of
Patients with Hepatitis C Virus of Various Genotypes
Acknowledgment: The authors thank Dr. Toshiro Tango
for the statistical analysis of the data.
Requests for Reprints: Makoto Mayumi, MD, Immunology
Division, Jichi Medical School, Tochigi-Ken 329-04, Japan.
Current Author Addresses: Drs. Akahane, Sakamoto,
and Miyazaki: The First Department of Internal Medicine,
Yamanashi Medical College, Yamanashi-Ken 409-38, Japan.
Dr. Kojima: Kojima Clinic, Gifu-Ken 500, Japan.
Dr. Sugai: Department of Internal Medicine, Iwaki Kyoritsu
General Hospital, Fukushima-Ken 973, Japan.
Mr. Tanaka: Japanese Red Cross Saitama Blood Center,
Saitama-Ken 362, Japan.
Dr. Tsuda: The Viral Hepatitis Research Foundation of Japan,
Tokyo 113, Japan.
Dr. Mishiro: Institute of Immunology, Tokyo 112, Japan.
Drs. Okamoto and Mayumi: Immunology Division, Jichi Medical
School, Tochigi-Ken 329-04, Japan.
Dr. Miyakawa: Mita Institute, Tokyo 108, Japan.
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