Prevalence of Gallbladder Disease among Persons with Hepatitis C Virus Infection in the

United States

MPH Capstone Paper

Edmund J. Bini, MD, FACP, FACG

Faculty Mentor: John McGready, MS

Edmund J. Bini, MD

ABSTRACT

Background: Gallstones are a major public health problem and this disorder is one of the most common and costly

of all digestive diseases. Although liver cirrhosis is a well-documented risk factor for the formation of gallstones,

little is known about the prevalence of gallbladder disease (GBD) in persons with hepatitis C virus (HCV) infection.

Aims: To determine the prevalence of GBD in persons with HCV infection in a representative sample of the general

adult population of the United States.

Study Design: Cross-sectional national survey.

Setting: The Third National Health and Nutrition Examination Survey (NHANES III), 1988 – 1994.

Participants: 13,465 persons 20 – 74 years of age for whom data on HCV infection and gallbladder

ultrasonography were complete.

Measurements: The presence of GBD was ascertained by abdominal ultrasonographic evidence of gallstones or

cholecystectomy. The presence of HCV infection was assessed by a positive HCV antibody test and a positive HCV

RNA test.

Results: Of the 13,465 persons evaluated, 1.6% (95% CI, 1.1% – 2.1%) had chronic HCV infection and 12.5%

(95% CI, 11.3% – 13.7%) had GBD. After adjusting for potential confounding variables, the relative odds of

gallstones (OR = 3.20; 95% CI 1.08 – 9.45; p = 0.036), cholecystectomy (OR = 4.57; 95% CI 1.57 – 13.27; p =

0.006), and GBD (OR = 3.86; 95% CI, 1.51 – 9.86; p = 0.006) among HCV positive men was significantly elevated

compared to HCV negative men. In contrast, the adjusted relative odds of gallstones (OR = 2.55; 95% CI, 0.58 –

11.25; p = 0.211), cholecystectomy (OR = 0.70; 95% CI, 0.21 – 2.37; p = 0.563), and GBD (OR = 1.59; 95% CI,

(0.54 – 4.64; p = 0.391) among HCV positive women was not significantly higher compared with HCV negative

women. The prevalence of GBD differed significantly according to HCV genotype (p = 0.028), with the prevalence

of GBD being highest in persons infected with genotype 2a and lowest in those with genotype 4. The relative odds

of GBD increased significantly with the severity of liver disease as assessed by elevated serum total bilirubin levels

and low levels of serum albumin and platelet counts.

Conclusions: In the United States, HCV infection is a major risk factor for GBD among men but not in women and

GBD was more common in those with severe liver disease.

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Edmund J. Bini, MD

Gallstones are a major public health problem and this disorder is one of the most common and

costly of all digestive diseases (1). The Third National Health and Nutrition Examination Survey

(NHANES III) estimated that 20.5 million persons in the United States had gallbladder disease (GBD)

(2). There are more than one thousand deaths per year from GBD in the United States, and the mortality

rate is 0.84 per 100,000 persons (1). Furthermore, the annual direct and indirect costs of GBD in the

United States in the year 2000 was approximately 6.5 billion dollars, and it is the second most costly

digestive disease (1).

Although liver cirrhosis is a well-documented risk factor for the formation of gallstones (3-21),

little is known about the prevalence of GBD in persons with hepatitis C virus (HCV) infection. In

addition to GBD, HCV infection is also an important public health problem in the United States and it is a

leading cause of chronic liver disease and cirrhosis (22, 23). Data from NHANES III estimated that

approximately 3.9 million people (1.8% of the population) have been infected with HCV in the United

States, and approximately 2.7 million people have chronic infection (24). Despite the high prevalence of

both GBD and HCV infection in the United States, no population-based studies of GBD have been

conducted among persons with HCV infection.

Ultrasonography of the gallbladder and HCV testing were both performed as part of NHANES

III, resulting in population-based estimates derived from approximately 14,000 participants (25). The

primary aim of this study was to determine the prevalence of GBD in persons with HCV infection in the

United States. We hypothesized that persons with HCV infection have a higher prevalence of GBD than

those without HCV infection after adjusting for important confounding variables.

METHODS

Study Population

NHANES III was conducted from 1988 – 1994 by the National Center for Health Statistics to

obtain national statistics on the health and nutritional status of the noninstitutionalized civilian population

of the United States by means of household interviews, as well as standardized physical examinations,

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Edmund J. Bini, MD

abdominal ultrasonography, and collection of blood samples in special mobile examination centers.

NHANES III included a sample of 33,994 persons at least two months of age at 89 randomly selected

locations throughout the United States (25). The study protocol was reviewed and approved by an

institutional review board at the Centers for Disease Control and Prevention and all participants provided

written informed consent.

NHANES III was based on a complex, stratified, multistage, probability-sample design (25).

Persons less than 5 years of age or 60 years of age or older, non-Hispanic blacks, and Mexican Americans

were sampled at higher frequencies than other persons. After weighting on the basis of age, sex,

race/ethnicity, and level of education, the distribution of participants was similar to that of the United

States population as a whole.

The target population for the gallbladder examination in NHANES III was the household

population of adults 20 – 74 years of age. A total of 16,115 persons 20 – 74 years of age were interviewed

at home, and 14,294 of these individuals completed the ultrasound examination of the gallbladder in the

mobile examination centers. We excluded 56 persons because of inability to properly visualize the

gallbladder lumen (9 with porcelain gallbladder and 47 because the gallbladder was not well seen),

leaving 14,238 with complete ultrasound examinations. Among these 14,238 persons, 13,495 individuals

had HCV antibody testing performed. After excluding 30 individuals with indeterminate HCV antibody

test results, the remaining 13,465 persons constitute the study sample (Figure 1).

Study Design

Ultrasound examinations were conducted with persons in both supine and left decubitus positions

with a Toshiba (Tustin, CA) SSA-90A machine using 3.75-MHz and 5.0-MHz transducers (25). A

diagnosis of gallstones required 2 views showing echoes within the gallbladder and echo shadowing. If a

right upper quadrant or epigastric scar was observed and the gallbladder was not seen, it was concluded

that a cholecystectomy had been performed.

All examinations were videotaped for an independent review by expert radiologists without

knowledge of the technician's diagnosis. When the diagnosis made by the ultrasonographer and

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Edmund J. Bini, MD

radiologist differed, the videotape was reviewed by a senior radiologist for final adjudication. Agreement

between examining ultrasonographers and videotape review by radiologists was 99% with a kappa

statistic of 0.94 for the diagnosis of GBD (2).

Presence of antibody to HCV was assessed by using a second-generation enzyme immunoassay

test (Abbott Laboratories, Chicago, Illinois). Positive specimens were tested in duplicate, and repeatedly

positive samples were tested again by using the HCV MATRIX assay (Abbott Laboratories). Specimens

that were positive according to the HCV MATRIX assay were considered to be HCV antibody positive

(24).

Testing for HCV RNA using reverse-transcriptase polymerase-chain-reaction amplification of the

5' noncoding region was performed on HCV antibody positive samples (24). Samples found to be

negative for HCV RNA were extracted a second time by the same procedure with an additional

incubation at 50 degrees Celsius for 45 minutes with 25 units of reverse transcriptase (Boehringer

Mannheim Diagnostics, Indianapolis, IN) and 10 units of RNAsin (Boehringer Mannheim Diagnostics).

The genotype of HCV RNA positive samples were determined by sequencing of 300 nucleotides in the

NS5b region (24). Tests for hepatitis B surface antigen were performed using a sandwich

radioimmunoassay (Abbott Laboratories).

In addition to abdominal ultrasonography and HCV testing, NHANES III collected information

on many other variables that could potentially confound the relationship between HCV infection and

GBD (2). For both men and women, potential covariates that were considered in these analyses included

age, race/ethnicity (non-Hispanic white, non-Hispanic black, Mexican-American, and other), body mass

index (weight in kilograms divided by the square of height in meters), waist to hip circumference ratio,

highest completed year of education, poverty income ratio, breakfast frequency (everyday, some days,

rarely, weekends only, or never), voluntary weight loss in the prior 12 months, monthly leisure physical

activity as measured by the self-reported frequencies of 9 activities (walking, running or jogging, dancing,

swimming, bicycling, gardening, lifting weights, calisthenics, and aerobics), serum total cholesterol

levels, presence of diabetes (determined by self-report of a physician diagnosis of diabetes), alcohol use

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Edmund J. Bini, MD

(never drinker, past drinker, less than 1 drink per day, 1 – 2 drinks per day, and more than 2 drinks per

day), and tobacco use (never smoker, past smoker, less than 1 pack per day, and at least 1 pack per day).

In addition to the potential confounding variables listed above, covariates that were included in the

analyses for women included the number of live births and duration of oral contraceptive use in months.

Study Outcomes

The primary outcome of this study was the prevalence and relative odds of GBD among HCV

positive persons compared to those without HCV infection. For the analysis of HCV status, persons were

considered HCV positive if they had a positive HCV antibody and were HCV RNA positive by

polymerase-chain-reaction. Persons were considered HCV negative if they were HCV antibody negative

or if they were HCV antibody positive but HCV RNA was not detected by polymerase-chain-reaction.

GBD was defined by the presence of one or more gallstones on ultrasound examination or if there was

evidence of surgical removal of the gallbladder.

Secondary outcomes of this study were the prevalence of GBD in HCV positive persons

according to the genotype of HCV infection, determination of the relative odds of GBD in HCV positive

persons according to the severity of liver disease, and the prevalence of GBD among hepatitis B virus

(HBV) positive subjects. The severity of liver disease was determined by serum total bilirubin levels,

serum albumin levels, platelet counts, and the aspartate aminotransferase (AST)/alanine aminotransferase

(ALT) ratio. Patients were considered HBV positive if they had hepatitis B surface antigen detected in

serum.

Statistical Analysis

General descriptive analysis was performed to compare participants with and those without HCV

infection. Using statistical techniques to account for the complex survey design of NHANES III,

categorical variables were compared using Pearson chi-square tests, while continuous variables were

compared using t-tests.

Because of sex differences in the prevalence of and risk factors for HCV infection and GBD in

the United States, we conducted separate analyses for men and women (2). Univariate and multivariate

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Edmund J. Bini, MD

complex survey logistic regression techniques were used to determine the sex-specific crude and adjusted

odds ratios (OR) and 95% confidence intervals (CI) of GBD with respect to HCV and HBV infection. All

statistical analyses were performed using Stata SE software, version 8.2 (Stata Corp., College Station,

TX). Sampling weights based on United States Census data describing age, sex, ethnicity, income, and

geographic location of the United States population were used in all analyses to provide accurate

estimates that were representative of the United States population during that time. These sampling

weights account for the unequal probabilities of selection as a result of the cluster design, non-response,

and the planned oversampling of certain ethnic minority populations (25).

RESULTS

Participant Characteristics

Antibodies to HCV were present in 2.3% (95% CI, 1.8% – 2.8%) of the 13,465 participants. The

prevalence of antibodies to HCV was 3.1% (95% CI, 2.3% – 4.0%) in men and 1.5% (95% CI, 1.1% –

1.9%) in women, and this difference was statistically significant (p = 0.001).

Among HCV antibody positive persons, 76.6% (95% CI, 66.9% – 84.2%) were HCV RNA

positive by polymerase-chain-reaction and, therefore, had chronic HCV infection with viremia. Overall,

1.6% (95% CI, 1.1% – 2.1%) of the population was HCV RNA positive, and the prevalence was

significantly higher in men (2.2%; 95% CI, 1.4% – 3.0%) than in women (1.0%; 95%, CI 0.6% – 1.5%; p

= 0.004).

The population characteristics stratified by HCV status are shown in Table 1. Compared to HCV

negative persons, participants who were HCV positive were significantly younger, were more likely to be

men, were less likely to be white, had a lower education and poverty income ratio, were less likely to

voluntarily try to lose weight in the past 12 months, had lower serum total cholesterol levels, consumed

more alcohol, and were more likely to smoke.

Overall Prevalence of GBD

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Edmund J. Bini, MD

The prevalence of GBD in the 13,465 persons in this study was 12.5% (95% CI, 11.3% – 13.7%),

including gallstones in 7.1% (95% CI, 6.3% – 7.9%) and cholecystectomy in 5.4% (95% CI, 4.8% –

5.9%). The overall prevalence of gallstones (8.6% [95% CI, 7.4% – 9.8%] vs. 5.6% [95% CI, 4.7% –

6.5%], p < 0.001), prior cholecystectomy (8.1% [95% CI, 7.2% – 9.0%] vs. 2.4% [95% CI, 1.8% – 3.1%],

p < 0.001), and GBD (16.7% [95% CI, 15.1% – 18.4%] vs. 8.0% [95% CI, 6.7% – 9.3%], p < 0.001) were

all significantly higher in women than in men.

GBD in HCV Positive Persons

Overall, there were approximately 404,847 (95% CI, 135,550 – 674,144) persons with chronic

HCV infection and GBD in the United States. The crude prevalence of gallstones, cholecystectomy, and

GBD (gallstones and cholecystectomy combined) among HCV positive persons is shown in Table 2. For

both men and women, the crude prevalence of GBD was higher in HCV positive persons than in those

who were HCV negative, although only the crude relative odds of gallstones in women was statistically

significant (OR = 2.84; 95% CI, 1.07 – 7.51; p = 0.036).

The prevalence of GBD increased with age among men with HCV and among men without HCV

(Figure 2A). The proportion of men with GBD was higher in HCV positive persons than in HCV

negative persons for all age groups except those 20 – 29 years of age. Among HCV positive women, the

highest prevalence of GBD was seen among those persons 50 – 59 years of age, while the prevalence of

GBD increased in a linear fashion with age among HCV negative women (Figure 2B). The proportion

with GBD was higher in HCV positive women than in HCV negative women for all age groups except

those 20 – 29 years of age.

The prevalence of GBD was higher in HCV positive men compared with HCV negative men

among non-Hispanic blacks and Mexican Americans (Figure 3A). In contrast, the prevalence of GBD

was similar in HCV positive and HCV negative non-Hispanic white men. The prevalence of GBD was

higher in HCV positive women compared with HCV negative women for non-Hispanic whites, non-

Hispanic blacks, and Mexican Americans (Figure 3B).

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Edmund J. Bini, MD

Potential risk factors that might confound the relationship between HCV and GBD were

evaluated in sex-specific multivariate logistic regression models (Table 2). After adjusting for potential

confounding variables, the relative odds of gallstones (OR = 3.20; 95% CI 1.08 – 9.45; p = 0.036),

cholecystectomy (OR = 4.57; 95% CI 1.57 – 13.27; p = 0.006), and GBD (OR = 3.86; 95% CI, 1.51 –

9.86; p = 0.006) among HCV positive men was significantly elevated compared to HCV negative men. In

contrast, the adjusted relative odds of gallstones, cholecystectomy, and GBD among HCV positive

women was not significantly higher compared to HCV negative women.

Prevalence of GBD According to HCV Genotype

Genotype was determined for 96.9% of the HCV positive samples and 56.2% were classified as

1a, 14.4% as 1b, 2.9% as 2a, 11.0% as 2b, 8.2% as 3a, 0.9% as 4, and 6.4% as genotype 6. The

prevalence of GBD stratified by HCV genotype is shown in Figure 4. The proportion of persons that had

GBD differed significantly according to genotype (p = 0.028), with the prevalence of GBD being highest

in persons infected with genotype 2a and lowest in those with genotype 4. However, there was no

statistically significant difference in the prevalence of GBD among persons with genotype 1 (17.1%; 95%

CI, 4.4% – 29.9%) compared to those with other genotypes (15.8%; 95% CI, 0.0% – 34.8%; p = 0.911).

GBD and the Severity of Liver Disease in HCV Positive Persons

In order to determine whether more severe liver disease was associated with a higher prevalence

of GBD among HCV infected persons, we evaluated the association between GBD and serum total

bilirubin levels, serum albumin levels, platelet counts, and the AST/ALT ratio (Table 3). The relative

odds of GBD increased with increasing levels of serum total bilirubin levels and this association remained

statistically significant after adjusting for age, gender, and race/ethnicity. In addition, lower serum

albumin levels and lower platelet counts were associated with an increased relative odds of GBD in both

unadjusted and adjusted multivariate logistic regression models. Although an AST/ALT ratio of > 1.00

was significantly associated with an increased relative odds of GBD in the unadjusted analysis, the

association did not persist after adjusting for age, sex, and race/ethnicity.

GBD in Hepatitis B Virus-Infected Persons

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Edmund J. Bini, MD

The prevalence of HBV infection among the study population was 0.45% (95% CI, 0.29% –

0.60%). The proportion of men with HBV infection (0.60%, 95% CI, 0.34% – 0.85%) was significantly

higher than the proportion of women infected with HBV (0.30%; 95% CI, 0.15% – 0.46%; p = 0.033).

There was no increase in the prevalence of GBD among men (OR = 0.26; 95% CI, 0.05 – 1.25; p = 0.092)

or women (OR = 0.47; 95% CI, 0.06 – 3.42; p = 0.445) with HBV infection compared to those without

HBV infection even after adjusting for all potential confounding variables.

DISCUSSION

Ultrasonography is a rapid, non-invasive method of imaging the gallbladder, and this technique

has contributed greatly to our understanding of the epidemiology of and risk factors for GBD (26). The

risk of GBD increases with age (26, 27), and the prevalence is as high as 25.3% in men and 33.1% in

women 60 – 74 years of age in the United States (2). Overall, the prevalence of GBD in the United States

is higher in women (16.6%) than in men (7.9%) between the ages of 20 – 74 years (2). Population-based

studies have revealed a marked variation in the prevalence of GBD among different racial/ethnic groups

(2, 28, 29). The age-standardized prevalence of GBD in the United States was similar for non-Hispanic

white men (8.6%) and Mexican American men (8.9%), and both were higher than in non-Hispanic black

men (5.3%). Among women, the age-adjusted prevalence was higher in Mexican Americans (26.7%) than

in non-Hispanic whites (16.6%) and non-Hispanic blacks (13.9%) (2), and American Indian and Hispanic

women are at particularly high-risk of developing GBD (28, 29).

In addition to age, sex, and race/ethnicity, other potential risk factors for GBD include obesity (2,

30-32), rapid weight loss (31), lower levels of physical activity (2, 33, 34), pregnancy (35-37), increasing

number of live births (2), oral contraceptive use and estrogen replacement therapy (38, 39), diabetes

mellitus (2, 40-42), abstinence from alcohol (2, 41, 43), smoking (2), low total serum cholesterol levels

(2, 15), low levels of coffee consumption (44, 45), and genetic factors (46, 47). However, some of these

variables have been inconsistently found to be associated with GBD and these risk factors may differ

considerably among men and women (2, 15).

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Edmund J. Bini, MD

Liver cirrhosis has also been shown to be an important risk factor for the development of GBD

(3-21). Although cholesterol stones are the most common type of gallstones in the general population,

pigment stones are the most prevalent type in cirrhotic patients (3, 6, 48). Several investigators have noted

that the risk of GBD varies according to the etiology of cirrhosis (9, 20). In a prospective study of 165

cirrhotic patients followed for a mean of 33 months, the incidence of new gallstones was 28.9% in

alcoholic cirrhosis but only 1.9% in those with cirrhosis from viral hepatitis (9). In contrast, Buchner and

Sonnenberg (20) found that the prevalence of gallstones was lower in patients with alcoholic cirrhosis

(9%) compared to those with non-alcoholic cirrhosis (14%).

In contrast to the abundance of literature on the association between cirrhosis and GBD, we are

unaware of any other population-based studies of the relationship between HCV infection and GBD. In a

retrospective review of 1,028 subjects undergoing a periodic health examination at China Medical

College Hospital in Taiwan, multivariate analysis identified HCV infection as a risk factor for GBD in

women (OR = 3.6; 95% CI, 1.4 – 9.7) but not in men (49). In contrast, O’Sullivan et al. (50) found that

the incidence of gallstones in patients infected with HCV was no different than the incidence of gallstones

in the general population in Ireland.

In the present study, we found that HCV infection was a strong risk factor for GBD in men (OR =

3.86; 95% CI, 1.51 – 9.86) but not in women (OR = 1.59; 95% CI, 0.54 – 4.64). The reason for the sex-

specific differences in the association between HCV infection and GBD are not known. However, these

findings are in agreement with other studies that have shown that cirrhosis is a risk factor for GBD in men

but not in women (5, 18). It is likely that the pathophysiology of and risk factors for gallstone formation

differ among men and women (2, 15). Alternatively, we may have failed to detect a statistically

significant association between HCV infection and GBD due to the small proportion of women with

chronic HCV (1.0%) or due to the fact that women already have a high prevalence of GBD even in the

absence of HCV infection.

The mechanisms leading to the development of gallstones in patients with cirrhosis of the liver

are not well understood. The increased risk of gallstone formation in cirrhotic patients, especially in those

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Edmund J. Bini, MD

with advanced liver disease, is likely multifactorial and proposed mechanisms include reduced hepatic

synthesis and transport of bile salts (51), impaired gallbladder motility (19, 52, 53), high estrogen levels

(17, 54), and chronic hemolysis secondary to hypersplenism (4, 6). Impaired gallbladder motility can

result in decreased gallbladder emptying in response to a meal, bile stasis, and increased gallstone

formation, and one study proposed that impaired gallbladder motility in cirrhotics with gallstones is due

to autonomic dysfunction (53). Although high estrogen levels have been suggested as a possible

mechanism of increased gallstone formation in cirrhotic patients, Li et al. (19) did not find any significant

differences in plasma levels of sex hormones (estradiol and testosterone) between cirrhotic patients with

and those without gallstones.

In addition to the potential mechanisms of increased gallstone formation in cirrhotic patients

described above, direct infection of the gallbladder by HCV may also play an important role in the

development of GBD. Loriot et al. (55) demonstrated that HCV can successfully infect gallbladder

epithelial cells. Although HCV replication in gallbladder epithelial cells was low, the results were highly

reproducible in that study. Other investigators have also detected HCV RNA and HCV antigens in

gallbladder specimens obtained from HCV-infected patients at the time of autopsy (56). It is possible that

HCV infection of the gallbladder may increase the risk of gallstone formation by causing altered

gallbladder mucosal function or gallbladder dysmotility, and further investigations to address this

interesting hypothesis are needed.

The present investigation found that the relative odds of GBD among persons with HCV infection

increased with the severity of liver disease as assessed by serum total bilirubin levels, serum albumin

levels, and platelet counts. Several other studies have also demonstrated that the prevalence and incidence

of GBD was significantly higher in patients with advanced cirrhosis compared to those with well-

compensated liver disease (4-6, 8-11, 13, 18, 19). In a prospective study of 165 cirrhotic patients, Fornari

et al. (9) noted that the cumulative incidence of gallstones at 48 months was 49.3% in patients with Child-

Pugh class C cirrhosis compared with 24.0% in patients with class B and 6.4% in those with class A

cirrhosis.

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Edmund J. Bini, MD

The risk of gallstones becoming symptomatic is higher in patients with cirrhosis due to viral

hepatitis than in those with alcoholic cirrhosis (21). In addition, patients with cirrhosis are more likely to

undergo cholecystectomy for emergent reasons than those without liver disease (57). These findings, as

well as the results of the present study showing a high prevalence of GBD in persons with advanced liver

disease in the United States, have important implications because cholecystectomy for symptomatic

gallstones in patients with advanced liver disease is associated with a high risk of morbidity and mortality

(57, 58).

After adjusting for potential confounding variables, the present study did not find a statistically

significant association between HBV infection and GBD. It is interesting to note that the adjusted relative

odds of GBD in HBV-infected men (OR = 0.26; 95% CI, 0.05 – 1.25) and women (OR = 0.47; 95% CI,

0.06 – 3.42) in the NHANES III study participants was less than 1, suggesting a protective effect of HBV

on the development of GBD. Although some investigators have shown that HBV was not a risk factor for

GBD (59-61), others reported that HBV infection was a strong risk factor for GBD (62). Interestingly,

one study of 603 patients with chronic hepatitis or cirrhosis found that gallstones were present in only

11.9% of HBV-infected patients compared with 36.2% of those without HBV, and also demonstrated that

HBV infection was protective for the development of gallstones in multivariate analysis (13).

The main strength of using the NHANES III data is that it included a large sample size and it

allowed for the determination of direct estimates of the prevalence of GBD among HCV-infected persons

in the United States population rather than relying on the prevalence in a highly selected population.

Other strengths of NHANES III included the collection of detailed data on risk factors for GBD, the use

of ultrasonography to make the diagnosis of GBD, and the availability of HCV RNA testing to diagnose

chronic HCV infection.

However, several limitations should be considered when interpreting our findings. Due to the

cross-sectional design of NHANES III, a temporal relationship between HCV infection and GBD could

not be established. In addition, some risk factors for GBD may have changed from the time when

gallstones formed and data were not available on some potential risk factors such as rapid weight loss.

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Edmund J. Bini, MD

Another potential limitation is that cholesterol and pigment stones were not differentiated in this study,

and, therefore, we cannot be sure that the observed differences between HCV positive and HCV negative

persons were due to differences in the risk of a particular type of gallstone. Finally, another limitation of

this study is the use of surrogate markers (serum total bilirubin, albumin, platelet count, and AST/ALT

ratio) to determine the severity of liver disease since liver biopsies were not performed.

In conclusion, our study demonstrates that chronic HCV infection is an important risk factor for

GBD in men but not in women in the United States. Among persons with HCV infection, the prevalence

of GBD is highest among those with more severe liver disease. The findings of this study are important

because of the large number of persons in the United States with HCV infection and GBD. In addition,

this study has important public health implications because cholecystectomy for symptomatic gallstones

in patients with advanced liver disease is associated with a high risk of morbidity and mortality. Further

research is needed to determine the temporal relationship between HCV infection and GBD, as well as the

incidence of symptomatic gallstones, the optimal management of GBD, and the outcomes of

cholecystectomy among persons with HCV. In addition, future studies to evaluate the impact of antiviral

therapy on the incidence of GBD among persons with HCV infection in the United States are warranted.

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beneficial and harmful influences of alcohol. Am J Gastroenterol 2002;97:905-909.

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cirrhosis: a case-control study. Am J Gastroenterol 2003;98:1856-1860.

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virus (HCV) infection and HCV-related chronic disease. MMWR Morb Mortal Wkly Rep 1998;47:1-39.

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Examination Survey, 1988-94. Vital Health Stat 1 1994;32:1-407.

26. Kratzer W, Mason RA, Kachele V. Prevalence of gallstones in sonographic surveys worldwide. J Clin

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the Sirmione Study. Hepatology 1987;7:913-917.

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28. Everhart JE, Yeh F, Lee ET et al. Prevalence of gallbladder disease in American Indian populations:

findings from the Strong Heart Study. Hepatology 2002;35:1507-1512.

29. Everhart JE. Gallstones and ethnicity in the Americas. J Assoc Acad Minor Phys 2001;12:137-143.

30. Ruhl CE, Everhart JE. Relationship of serum leptin concentration and other measures of adiposity with

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31. Everhart JE. Contributions of obesity and weight loss to gallstone disease. Ann Intern Med 1993;119:1029-

1035.

32. Maclure KM, Hayes KC, Colditz GA et al. Weight, diet, and the risk of symptomatic gallstones in middle-

aged women. N Engl J Med 1989;321:563-569.

33. Leitzmann MF, Giovannucci EL, Rimm EB et al. The relation of physical activity to risk for symptomatic

gallstone disease in men. Ann Intern Med 1998;128:417-425.

34. Leitzmann MF, Rimm EB, Willett WC et al. Recreational physical activity and the risk of cholecystectomy

in women. N Engl J Med 1999;341:777-784.

35. Thijs C, Knipschild P, Leffers P. Pregnancy and gallstone disease: an empiric demonstration of the

importance of specification of risk periods. Am J Epidemiol 1991;134:186-195.

36. Maringhini A, Ciambra M, Baccelliere P et al. Biliary sludge and gallstones in pregnancy: incidence, risk

factors, and natural history. Ann Intern Med 1993;119:116-120.

37. Everson GT. Pregnancy and gallstones. Hepatology 1993;17:159-161.

38. Uhler ML, Marks JW, Judd HL. Estrogen replacement therapy and gallbladder disease in postmenopausal

women. Menopause 2000;7:162-167.

39. Thijs C, Knipschild P. Oral contraceptives and the risk of gallbladder disease: a meta-analysis. Am J Public

Health 1993;83:1113-1120.

40. Ruhl CE, Everhart JE. Association of diabetes, serum insulin, and C-peptide with gallbladder disease.

Hepatology 2000;31:299-303.

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approach to the prevention of gallstones? Am J Public Health 1987;77:841-845.

42. Haffner SM, Diehl AK, Mitchell BD et al. Increased prevalence of clinical gallbladder disease in subjects

with non-insulin-dependent diabetes mellitus. Am J Epidemiol 1990;132:327-335.

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43. Leitzmann MF, Tsai CJ, Stampfer MJ et al. Alcohol consumption in relation to risk of cholecystectomy in

women. Am J Clin Nutr 2003;78:339-347.

44. Leitzmann MF, Willett WC, Rimm EB et al. A prospective study of coffee consumption and the risk of

symptomatic gallstone disease in men. JAMA 1999;281:2106-2112.

45. Leitzmann MF, Stampfer MJ, Willett WC et al. Coffee intake is associated with lower risk of symptomatic

gallstone disease in women. Gastroenterology 2002;123:1823-1830.

46. Gilat T, Feldman C, Halpern Z et al. An increased familial frequency of gallstones. Gastroenterology

1983;84:242-246.

47. Sarin SK, Negi VS, Dewan R et al. High familial prevalence of gallstones in the first-degree relatives of

gallstone patients. Hepatology 1995;22:138-141.

48. Diehl AK, Schwesinger WH, Holleman DR, Jr. et al. Clinical correlates of gallstone composition:

distinguishing pigment from cholesterol stones. Am J Gastroenterol 1995;90:967-972.

49. Lai SW, Ng KC. Risk factors for gallstone disease in a hospital-based study. South Med J 2002;95:1419-

1423.

50. O'Sullivan MJ, Evoy D, O'Donnell C et al. Gallstones and laparoscopic cholecystectomy in hepatitis C

patients. Ir Med J 2001;94:114-117.

51. Alvaro D, Angelico M, Gandin C et al. Physico-chemical factors predisposing to pigment gallstone

formation in liver cirrhosis. J Hepatol 1990;10:228-234.

52. Acalovschi M, Dumitrascu DL, Csakany I. Gastric and gall bladder emptying of a mixed meal are not

coordinated in liver cirrhosis--a simultaneous sonographic study. Gut 1997;40:412-417.

53. Chawla A, Puthumana L, Thuluvath PJ. Autonomic dysfunction and cholelithiasis in patients with

cirrhosis. Dig Dis Sci 2001;46:495-498.

54. Stauber RE, Rosenblum E, Eagon PK et al. The effect of portal-systemic shunting on hepatic sex hormone

receptors in male rats. Gastroenterology 1991;100:168-174.

55. Loriot MA, Bronowicki JP, Lagorce D et al. Permissiveness of human biliary epithelial cells to infection by

hepatitis C virus. Hepatology 1999;29:1587-1595.

56. Yan FM, Chen AS, Hao F et al. Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis

C. World J Gastroenterol 2000;6:805-811.

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57. Puggioni A, Wong LL. A metaanalysis of laparoscopic cholecystectomy in patients with cirrhosis. J Am

Coll Surg 2003;197:921-926.

58. Aranha GV, Sontag SJ, Greenlee HB. Cholecystectomy in cirrhotic patients: a formidable operation. Am J

Surg 1982;143:55-60.

59. Lu SN, Chang WY, Wang LY et al. Risk factors for gallstones among Chinese in Taiwan. A community

sonographic survey. J Clin Gastroenterol 1990;12:542-546.

60. Chen CY, Lu CL, Lee PC et al. The risk factors for gallstone disease among senior citizens: an Oriental

study. Hepatogastroenterology 1999;46:1607-1612.

61. Chen CY, Lu CL, Huang YS et al. Age is one of the risk factors in developing gallstone disease in Taiwan.

Age Ageing 1998;27:437-441.

62. Sheen IS, Liaw YF. The prevalence and incidence of cholecystolithiasis in patients with chronic liver

diseases: a prospective study. Hepatology 1989;9:538-540.

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Edmund J. Bini, MD

Table 1. Characteristics of the 13,465 Participants Stratified by HCV Status*

HCV Positive HCV Negative P-Value Age 39.1 ± 0.99 42.4 ± 0.36 0.003 Men Women

66.7% 33.3%

48.3% 51.7%

0.001

Race/Ethnicity Non-Hispanic white Non-Hispanic black Mexican American Other

58.3% 22.2% 6.9%

12.6%

76.6% 10.4% 5.3% 7.8%

0.001

Body mass index (kg/m2) 25.7 ± 0.55 26.6 ± 0.12 0.096 Waist to hip ratio 0.92 ± 0.007 0.91 ± 0.002 0.074 Highest year of education completed 11.1 ± 0.25 12.5 ± 0.09 < 0.001 Poverty income ratio† 1.8 ± 0.17 3.1 ± 0.06 < 0.001 Breakfast frequency Everyday Some days Rarely Weekends Never

35.4% 29.3% 16.9% 6.7%

11.6%

49.7% 22.5% 15.5% 6.4% 5.9%

0.115

Voluntary weight loss in last 12 months 24.3% 42.5% < 0.001 Number of leisure physical activities in last month 23.2 ± 4.3 21.2 ± 0.59 0.647 Serum total cholesterol level (mg/dL) 181.3 ± 4.3 203.6 ± 0.81 < 0.001 Physician-diagnosed diabetes 4.3% 4.9% 0.720 Alcohol use Never drinker Past drinker Less than 1 drink per day 1 – 2 drinks per day More than 2 drinks per day

6.7%

23.4% 23.8% 19.7% 26.3%

12.1% 31.7% 41.0% 8.8% 6.4%

< 0.001

Smoking status Never smoker Past smoker Less than 1 pack per day 1 or more packs per day

19.1% 16.8% 24.8% 39.3%

45.5% 25.6% 12.6% 16.4%

< 0.001

Number of live births‡ 2.5 ± 0.23 2.5 ± 0.04 0.810 Duration of oral contraceptive use in months‡ 30.8 ± 6.8 35.3 ± 1.3 0.504 *Continuous variables are expressed as mean ± standard error. Categorical variables are expressed as percentages;

actual numbers are not shown because the percentages incorporate sampling weights to account for the complex

survey design.

†The poverty income ratio was based on self-report of family income, family size, and tables published annually by

the U.S. Census Bureau. A poverty income ratio below 1.0 indicates that the person was below the poverty level.

‡Estimates are for women only.

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Edmund J. Bini, MD

Table 2. Relative Odds of Gallbladder Disease among HCV Positive Persons Compared to HCV Negative

Persons

Crude Prevalence (95% CI) Odds Ratios (95% CI)*

HCV Positive HCV Negative Crude Fully Adjusted†

Men

Gallstones

Cholecystectomy

Gallbladder Disease‡

7.5% (0.0% – 15.1%)

3.0% (0.0% – 7.3%)

10.5% (0.4% – 20.7%)

5.5% (4.6% – 6.4%)

2.4% (1.8% – 3.1%)

7.9% (6.7% – 9.2%)

1.40 (0.46 – 4.29)

1.23 (0.30 – 5.11)

1.36 (0.47 – 3.97)

3.20 (1.08 – 9.45)

4.57 (1.57 – 13.27)

3.86 (1.51 – 9.86)

Women

Gallstones

Cholecystectomy

Gallbladder Disease‡

20.8% (5.0% – 36.5%)

7.1% (1.2% – 13.0%)

27.9% (13.6% – 42.1%)

8.5% (7.2% – 9.7%)

8.1% (7.2% - 9.0%)

16.6% (15.0% – 18.3%)

2.84 (1.07 – 7.51)

0.86 (0.35 – 2.12)

1.94 (0.94 – 4.01)

2.55 (0.58 – 11.25)

0.70 (0.21 – 2.37)

1.59 (0.54 – 4.64)

*Odds ratios are for the comparison of HCV positive persons to those that were HCV negative.

†For both men and women, fully adjusted odds ratios were adjusted for age, race/ethnicity, body mass index, waist

to hip circumference ratio, highest completed year of education, poverty income ratio, breakfast frequency,

voluntary weight loss in the prior 12 months, monthly leisure physical activity, serum total cholesterol, presence of

diabetes, alcohol use, and tobacco use. In addition, the fully adjusted odds ratios for women were also adjusted for

the number of live births and duration of oral contraceptive use.

‡Gallbladder disease is defined as the presence of gallstones or evidence of cholecystectomy.

21

Edmund J. Bini, MD

Table 3. Relative Odds of Gallbladder Disease among HCV Positive Persons According to the Severity of

Liver Disease*

Crude Adjusted†

OR (95% CI) P-Value OR (95% CI) P-Value

Serum total bilirubin

≤ 1.0 mg/dL

1.1 – 2.0 mg/dL

> 2.0 mg/dL

Serum albumin

> 3.4 g/dL

3.1 – 3.4 g/dL

≤ 3.0 g/dL

Platelet count

≥ 200 x 106/µL

100 – 199 x 106/µL

< 100 x 106/µL

AST/ALT ratio

≤ 1.00

> 1.00

1.00 (reference)

1.32 (0.39 – 4.50)

39.79 (7.50 – 211.14)

1.00 (reference)

26.85 (6.90 – 104.49)

17.88 (3.12 – 102.30)

1.00 (reference)

3.28 (0.76 – 14.23)

42.74 (7.62 – 239.62)

1.00 (reference)

3.69 (1.10 – 12.44)

--

0.652

< 0.001

--

< 0.001

0.002

--

0.110

< 0.001

--

0.035

1.00 (reference)

1.04 (0.18 – 5.82)

33.48 (4.99 – 224.73)

1.00 (reference)

32.87 (4.46 – 242. 08)

22.59 (2.77 – 184.07)

1.00 (reference)

3.83 (0.65 – 22.56)

31.32 (2.35 – 416.71)

1.00 (reference)

2.42 (0.87 – 6.74)

--

0.966

0.001

--

0.001

0.004

--

0.135

0.010

--

0.089

*The severity of liver disease was assessed by serum levels of total bilirubin (reference range, ≤ 1.0 mg/dL), serum

albumin (reference range, 3.4 – 5.0 g/dL), platelet counts (reference range, 200 – 400 x 106/µL), and the aspartate

aminotransferase (AST)/alanine aminotransferase (ALT) ratio.

† Adjusted for age, sex, and race/ethnicity.

22

Edmund J. Bini, MD

Figure 1. Determination of the Study Sample

33,994 Persons Interviewed

16,115 20 – 74 Years of Age

14,294 Ultrasonography Performed

14,238 Valid Ultrasound Exam

13,495 HCV Testing Performed

13,465 Final Study Sample

23

Edmund J. Bini, MD

Figure 2A. Prevalence of Gallbladder Disease among HCV Positive and HCV Negative Men Stratified by Age

1.1%

8.1%

17.9% 16.5%

45.7%

1.3%

7.1%11.9%

24.8%

1.7%0.0%5.0%

10.0%15.0%20.0%25.0%30.0%35.0%40.0%45.0%50.0%

20 - 29 30 - 39 40 - 49 50 - 59 60 - 74

Age in Years

Prev

alen

ce o

f GBD

HCV PositiveHCV Negative

Figure 2B. Prevalence of Gallbladder Disease among HCV Positive and HCV Negative Women Stratified by

Age

28.4%

73.4%

50.7%

2.1%

12.7%6.6%

15.6%

24.4%

32.8%

10.3%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

20 - 29 30 - 39 40 - 49 50 - 59 60 - 74

Age in Years

Prev

alen

ce o

f GBD

HCV PositiveHCV Negative

24

Edmund J. Bini, MD

Figure 3A. Prevalence of Gallbladder Disease among HCV Positive and HCV Negative Men Stratified by

Race/Ethnicity

8.4%7.5% 7.5%

8.7%

5.3%4.2%

0.0%1.0%2.0%3.0%4.0%5.0%6.0%7.0%8.0%9.0%

10.0%

Whites (Non-Hispanic)

Blacks (Non-Hispanic)

MexicanAmericans

Prev

alen

ce o

f GB

D

HCV PositiveHCV Negative

Figure 3B. Prevalence of Gallbladder Disease among HCV Positive and HCV Negative Women Stratified by

Race/Ethnicity

20.0%

30.4% 29.2%

17.3%

21.8%

12.2%

0.0%

5.0%

10.0%

15.0%

20.0%

25.0%

30.0%

35.0%

Whites (Non-Hispanic)

Blacks (Non-Hispanic)

MexicanAmericans

Prev

alen

ce o

f GB

D

HCV PositiveHCV Negative

25

Edmund J. Bini, MD

Figure 4. Prevalence of Gallbladder Disease Stratified by HCV Genotype

p = 0.028 by Pearson chi-square analysis

15.8%22.3%

76.8%

1.9% 1.5% 0.0%

32.2%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

1a 1b 2a 2b 3a 4 6

HCV Genotype

Prev

alen

ce o

f GB

D

26