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Hepatology Research 36 (2006) 100–106

Routes of infection and clinical outcome of Mexican women


reactive to anti-hepatitis C virus antibodies
Erwin Chiquete a,b , Laura V. Sánchez a,b , Arturo Panduro a,b,∗
aDepartment of Molecular Biology in Medicine, Civil Hospital of Guadalajara “Fray Antonio Alcalde”,
Hospital 278, Guadalajara, Postal Code 44280, Jalisco, Mexico
b Department of Physiology, University Center of Health Sciences (CUCS), University of Guadalajara, Guadalajara, Jalisco, Mexico

Received 27 January 2006; received in revised form 12 June 2006; accepted 15 June 2006
Available online 1 August 2006

Abstract

Background: Risk factors for the hepatitis C virus (HCV) infection influence both the frequency and the progression of the liver disease.
Routes of transmission and severity of the liver damage may differ by gender. We aimed to describe the risk factors for HCV infection and
for the severity of the liver disease among women seroreactive to anti-HCV antibodies. Also, we tested the hypothesis that length of infection
influences the levels of anti-HCV, in transfusion-associated hepatitis C.
Methods: Eighty-six interferon-naive women, repeatedly seroreactive to anti-HCV antibodies, aged > 20 years, were studied.
Results: Surgeries (80%) and transfusion before 1993 (58%) were the main risk factors (52% cases had both). The main reason for practicing
surgery was obstetric/gynecologic (74%). The main indication for transfusion was also obstetric/gynecologic (68%). Fifty-five (64%) women
were positive to HCV RNA in serum, of them, coinfection with the hepatitis B virus (HBV) occurred in three (5%) cases, being occult
hepatitis B (i.e., positive to HBV DNA, but negative to hepatitis B surface antigen) in two (4%). In multivariate analysis, determining factors
of cirrhosis at histologic examination were age and the antecedent of transfusion before 1993. Anti-HCV levels correlated with the elapsed
time from transfusion to diagnosis, but not with age.
Conclusion: An obstetric/gynecologic indication was the most frequent reason for both surgery and transfusion. Hepatitis B coinfection had
a low prevalence and did not influence the severity of the liver disease, as age and the antecedent of transfusion certainly did. Infection length
influenced the levels of anti-HCV antibodies in transfusion-associated hepatitis C.
© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cirrhosis; Hepatitis B; Hepatitis C; Liver; Serology; Test; Transfusion

1. Introduction tion of HCV [1,9]. Nevertheless, little is known about the


indication for blood transfusion in women who underwent
In countries in which the use of illicit intravenous drugs this procedure before the screening programs [10] and the
is not frequent, the recipients of transfusion before the sys- relation of this antecedent with the progression of the liver
tematic screening for the hepatitis C virus (HCV) in blood disease.
banks represent the largest group of persons with the infec- The severity of the liver fibrosis in HCV infection has
tion [1–5]. Several risk factors for HCV infection and for been associated with some clinical antecedents, like mode
the progression of the liver disease may differ by gender of transmission [6,11–13], length of infection [6,7,13], age,
[6–10]. Compared with men, in some countries women have gender, alcohol consumption, body mass index [6,7], hepati-
transfusion of blood products as the main route of acquisi- tis B virus (HBV) coinfection [14], and other factors. Also,
different levels of anti-HCV antibodies are observed among
∗ Corresponding author. Tel.: +52 33 3614 7743; fax: +52 33 3614 7743. people with distinct clinical outcomes [5,15]; however, the
E-mail address: apanduro@prodigy.net.mx (A. Panduro). exact significance of this difference is scarcely known.

1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.hepres.2006.06.012
E. Chiquete et al. / Hepatology Research 36 (2006) 100–106 101

In some populations, women appear to be not only the Diagnostics, Chicago, IL, USA) was used in all sera sam-
most affected gender with HCV infection [5,10,16–18], but ples, first stored at −70 ◦ C. Immunoassay signal strength of
also a homogeneous group with respect to risk factors and the sample to cut-off rate (S/CO) is the numeric value of the
clinical outcome [10,19,20]. We aimed to describe the risk assay, which is directly proportional to the quantity of anti-
factors associated with HCV infection and events related to bodies against HCV [15,23–26]. S/CO ratio > 1 is considered
blood transfusion prior to the screening program for HCV a reactive (i.e., positive) result, according to the manufacturer.
in Mexican blood banks, in women currently seroreactive to
anti-HCV antibodies. Also, we sought to test the hypothesis 2.3. Detection of HCV RNA in serum
that the mode of transmission and the length of infection
influence the clinical outcome and anti-HCV antibodies A home-made, qualitative nested reverse-transcription
levels. polymerase chain reaction (RT-PCR) was used to detect
HCV RNA in all sera samples, first stored at −70 ◦ C. Total
RNA was extracted from each serum without pooling, using
2. Methods QIAamp Viral RNA Mini Kit (QIAGEN, Chatsworth, CA) as
indicated the manufacturer. Afterwards, RT was carried out
This study was performed from March 2002 to June 2004 to obtain complementary DNA (cDNA) using M-MLV RT
at the Department of Molecular Biology in Medicine, Hospi- kit (MMLV, GIBCO/BRL). PCR amplification of cDNA and
tal Civil de Guadalajara “Fray Antonio Alcalde”. The inter- later a nested-PCR were performed with two pairs of primers
nal Committee of Ethics of our hospital approved the present that hybridize in a segment of the 5 non-coding region of
study. Informed consent was obtained from all patients. HCV genome, as is described elsewhere [27]. Appropriate
measures were taken to minimize the risk of sample cross-
2.1. Study population contamination. These measures comprised the inclusion of
serum samples from normal subjects and aliquots of water as
Ambulatory patients were referred after our request from negative controls. Qualitative nested RT-PCR was performed
blood bank and departments of infectology, gastroenterol- per duplicate in all samples. Cases with detectable HCV RNA
ogy, general internal medicine and hematology to confirm in serum were referred to an external party to undergo liver
HCV infection after at least two screening tests for anti- biopsy, if the patient accepted this procedure.
HCV antibodies yielding reactive results. None of the patients
were referred to our research facility by the departments of 2.4. Assessment of HBV coinfection
obstetrics or gynecology. All patients had known their screen-
ing test status before arrival to our department. Consecutive Infection with HBV was investigated by detection of the
women older than 20 years and never treated for HCV infec- hepatitis B surface antigen (HBsAg) and anti-hepatitis B
tion were eligible for the study. In cases with transfusion of core antigen (anti-HBc) antibodies, using an automated third-
blood products, only those occurring before 1993 were con- generation microparticle enzyme immunoassay (MEIA, IMx
sidered at significant risk for HCV infection, since it was Version 3.0 Abbott Diagnostics, Chicago, IL, USA), as well
the year in which blood bank screening for anti-HCV anti- as by detection of HBV DNA using a home-made qualitative
bodies was introduced in Mexico [21,22]. In women who nested PCR. For HBV DNA assessment, an aliquot of 200 ␮l
received blood products in more than one occasion, the ear- of sera samples were obtained to isolate viral DNA using the
liest transfusion was considered as the putative source of the QIAmp Blood Kit (Qiagen, Chatsworth, CA). HBV DNA
infection. Decompensated cirrhosis was considered to occur was amplified by standardized first-round and nested PCR of
in a patient if jaundice, ascitis or collateral superficial veins S-gene fragment using the primers and conditions described
were present. Also, the antecedent of upper gastrointestinal previously [28]. PCR assay was practiced per duplicate in
bleeding, hepatic encephalopathy, tense ascitis, and sponta- all patients. The criterion for adjudication of a confirmed
neous peritonitis was considered as being part of the liver diagnosis and the measures taken to minimize the risk of
failure syndrome. Patients were excluded if a new assay for cross-contamination were the same as in the assessment of
anti-HCV performed in our laboratory tested negative or if HCV RNA. Reactive sera to HBV DNA undergone direct
complete history and clinical data were not available. sequencing to determine HBV genotypes, as is described
elsewhere [28].
2.2. Detection of anti-HCV antibodies
2.5. Data analysis
A new determination of anti-HCV was practiced in our
laboratory for all patients, in order to confirm the screen- Chi-square or Fisher exact test was used to assess nominal
ing tests performed prior to the arrival to our depart- variables in bivariate analyses, as corresponded. To compare
ment and to assess the quantity of anti-HCV antibodies. A quantitative variables between two groups, Student t-test and
semi-quantitative automated third-generation microparticle Mann–Whitney U-test were performed for parametric and
enzyme immunoassay (MEIA, IMx HCV Version 3.0 Abbott non-parametric variables, respectively. Pearson correlation
102 E. Chiquete et al. / Hepatology Research 36 (2006) 100–106

Table 1
Demographic and clinical characteristics of the 86 women analyzed
Variable Total HCV RNA-positive (n = 55) HCV RNA-negative (n = 31) P-valuea
Age
Median (range) 53 (21–82) 54 (21–79) 51 (22–82) 0.18
School education
More than 6 years, n (%) 68 (79) 42 (76) 26 (84) 0.58
Marital status
Married, n (%) 64 (74) 41 (74) 23 (74) 0.98
Single, n (%) 10 (12) 4 (7) 6 (19) 0.11
Divorced, n (%) 7 (8) 6 (11) 1 (3) 0.23
Widower, n (%) 5 (6) 4 (7) 1 (3) 0.45
Occupation
Home (i.e., housewives), n (%) 43 (50) 31 (56) 12 (39) 0.17
Liver enzymes activity
ALT, mean (S.D.) 53.1 (48.8) 64.2 (63.6) 39.8 (15.7) 0.25
AST, mean (S.D.) 49.2 (39.3) 59.4 (47.9) 35.9 (18.9) 0.16
Risk factors for HCV infection
Major surgeries, n (%)b 69 (80) 46 (84) 23 (74) 0.40
Blood transfusion, n (%)c 50 (58) 34 (62) 16 (52) 0.37
None identified, n (%) 12 (14) 6 (11) 6 (19) 0.34
ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCV, hepatitis C virus; NA, not applicable; S.D., standard deviation.
a P-value for differences between HCV RNA-positive and HCV RNA-negative women; Chi-square, Fisher exact test, t-test or Mann–Whitney U-test, as

corresponded.
b Any major surgical procedure before the first screening test for HCV infection yielded a reactive result.
c Transfusion of blood products before 1993.

was used in continuous distributions. To find independent pre- Hosmer–Lemeshow goodness-of-fit test, which was consid-
dictors of the presence of cirrhosis at histologic assessment, ered as reliable if P > 0.20. Final statistical analyses were
we did a multivariate analysis by a binary logistic regres- regarded as significant when P < 0.05. All P-values reported
sion model. Independent variables were chosen if P < 0.1 in are two-sided. SPSS v12.0 statistical package was used for
selection step by bivariate analysis, subsequently a forward- all calculations.
stepwise method was performed. Adjusted odds ratios (OR)
with 95% confidence intervals (CI) resulting in final step of
the model are provided. Corrected OR were calculated for 3. Results
categorical predictors in order to approximate to the exact rel-
ative risk in a small sample with a high frequency of a research In the period comprised by this study, 103 women
outcome, with the formula proposed by Zhang and Yu [29] aged > 20 years who tested reactive to anti-HCV antibodies
as follows: corrected OR = multivariate OR/[(1 − incidence were studied. Of them, nine cases were excluded because
of the outcome in the nonexposed group) + (incidence of the a new test for anti-HCV tested negative and eight cases
outcome in the nonexposed group × multivariate OR)]. The because complete data on medical records were not available.
fitness of the regression model was evaluated by using the Thus, 86 interferon-naive women, older than 20 years and

Table 2
Characteristics of the earliest transfusion in the 50 women who were recipients of blood products before 1993
Variable Total HCV RNA-positive (n = 34) HCV RNA-negative (n = 16) P-valuea
Indication for transfusion
Obstetric/gynecologic, n (%) 34 (68) 24 (71) 10 (62) 0.75
Anemia, n (%) 7 (14) 3 (9) 4 (25) 0.19
Major surgery, n (%)b 5 (10) 4 (12) 1 (6) 0.98
Injury, n (%)c 4 (8) 3 (9) 1 (6) 0.75
Time from transfusion to diagnosis
Median (range) in years 21 (7–39) 23 (7–39) 20 (9–33) 0.11
HCV, hepatitis C virus.
a P-value for differences between HCV RNA-positive and HCV RNA-negative women; Chi-square, Fisher exact test or Mann–Whitney U-test, as corre-

sponded.
b Major surgeries related to blood loss that required transfusion, before the first screening test for HCV infection yielded a reactive result.
c Injuries related to blood loss that required transfusion.
E. Chiquete et al. / Hepatology Research 36 (2006) 100–106 103

Table 3
Multivariate analysis on factors predicting the presence of cirrhosis in histologic preparations of the liver (36 biopsy specimens of HCV RNA-positive cases)a
Variable Regression coefficient S.E. OR (95% CI) Corrected OR (95% CI) P-value
Age 0.194 0.075 1.21 (1.05–1.41) NA 0.01
Transfusionb
0 = absent
1 = present 3.100 1.173 2.23 (1.20–221.14) 1.69 (1.14–3.80) 0.008
Constant −11.214 4.328 NA NA <0.001
CI, confidence interval; HCV, hepatitis C virus; NA, not applicable; OR, odds ratio; S.E., standard error.
a Adjusted for HBV coinfection, diabetes mellitus, and for anti-HCV antibodies levels. Hosmer–Lemeshow test for goodness of fit in final step of the

regression model: χ2 = 4.70, 7 d.f., P = 0.70.


b The antecedent of transfusion of blood products before 1993.

repeatedly reactive to anti-HCV antibodies were analyzed 3.2. Coinfection with HBV
for the purposes of this study (Table 1). Mean age was 50.4
years (median 53, range 21–82 years). Of the 86 women Three (3%) cases were positive to HBV DNA, two cor-
studied, 55 (64%) had detectable HCV RNA in serum responding to genotype H and one to genotype A. No HCV
(Table 1). RNA-negative cases with a positive result to HBV DNA in
serum were identified. Hence, confirmed coinfection (i.e., the
presence of both HCV and HBV genomes in serum) occurred
3.1. Risk factors for HCV infection in 3 (5%) of the 55 women who tested positive to HCV RNA.
Occult hepatitis B (i.e., seronegative to HBsAg, but positive
Any major surgical procedure and transfusion of blood to HBV DNA) occurred in two (4%) cases. One patient with
products before 1993 were the most frequent risk factors, coinfection had transfusion of blood products and surgeries
with 74 (86%) cases having one or another, and 45 (52%) as risk factors, the other two had only surgeries.
having both (Table 1). The main reason for practicing surgery
was obstetric/gynecologic (74%). There were no patients
who declared past or current illicit intravenous drug use, 3.3. Factors influencing liver disease
the antecedent of a sex partner infected with HCV or sex-
ual promiscuity. Twelve (14%) women had no identifiable Patients declaring significant alcohol intake (i.e., >10 g
risk factor for HCV infection. per day) were not identified. Thirty-six (42%) women had
decompensated liver cirrhosis at inclusion in the study; 30
(83%) of them with detectable HCV RNA in serum. Among
3.1.1. The antecedent of transfusion the 50 patients with the antecedent of transfusion, decom-
Median age of the 50 (58%) women with the antecedent pensated cirrhosis was more frequent in those who had >20
of transfusion was 54.5 years (range 21–82), which was dif- years of age at the moment of the transfusion (22 of 24, 92%)
ferent from the median of 46 years (range 22–72) in the 36 than in younger women (17 of 26, 65%) (P = 0.02). Among
women without this antecedent (P = 0.009). The most fre- the 55 women with detectable HCV RNA in serum, 36 (65%)
quent indication for transfusion was an obstetric/gynecologic underwent liver biopsy and histologic examination, with 13
complication (e.g., miscarriage, abruptio placentae, dysfunc- (36%) cases having chronic liver inflammation with fibrosis
tional uterine bleeding, and other conditions) in 34 (68%) and 23 (64%) cirrhosis. There were no cases with hepato-
cases, followed by anemia in 7 (14%) (Table 2). The median cellular carcinoma. In a multivariate analysis adjusted for
year of the earliest transfusion was 1980 (range 1968–1992). diabetes mellitus, HBV coinfection, HBV genotypes and for
The median time from the earliest transfusion to the diagno- anti-HCV antibodies levels; determining factors of cirrho-
sis of HCV infection by a reactive screening test was 21 years sis at histologic assessment were age and the antecedent of
(Table 2). transfusion (Table 3).

Table 4
Median (minimum and maximum) of anti-HCV antibodies levels, expressed as sample-to-cut-off ratio (S/CO), in relation to several characteristics
HCV RNAa (n = 86) Transfusionb (n = 86) Surgeriesc (n = 86)
Positive (n = 55) Negative (n = 31) Positive (n = 50) Negative (n = 36) Positive (n = 69) Negative (n = 17)
43.3 (1.6–146.5) 2.0 (1–65.7) 41.1 (1–146.5) 34.9 (1.1–67.4) 40.0 (1–146.5) 38.0 (1.1–58.1)
a P < 0.001, for differences between HCV RNA-positive and HCV RNA-negative women; Mann–Whitney U-test.
b P = 0.08, for differences between women with and without transfusion of blood products before 1993; Mann–Whitney U-test.
c P = 0.36, for differences between women with and without surgeries; Mann–Whitney U-test.
104 E. Chiquete et al. / Hepatology Research 36 (2006) 100–106

Fig. 1. Median and interquartile range of anti-HCV antibodies levels (expressed as sample-to-cut-off [S/CO] ratio) in different subgroups. Error bars indicate
minimum and maximum, excluding two extreme cases. (A) Women with or without transfusion of blood products before 1993. (B) Women with or without a
positive result to HCV RNA in serum. (C) Women with the antecedent of transfusion who had or had not detectable HCV RNA in serum. (D) Women without
the antecedent of transfusion who had or had not detectable HCV RNA in serum.

3.4. Levels of anti-HCV antibodies found that the antecedent of surgeries was the most preva-
lent risk factor for HCV infection, followed by transfusion
Anti-HCV antibodies levels were higher in patients of blood products before 1993. However, a great propor-
with decompensated cirrhosis than in asymptomatic women tion of patients who had the antecedent of surgeries also had
(median S/CO value 41.2 versus 36.2, respectively; P = 0.03). transfusion, in most cases with an obstetric or gynecologic
However, after considering only HCV RNA-positive cases indication. This finding had only been reported in a female
(n = 55), anti-HCV levels were not different between patients population from Ireland [10]. That most women with surg-
with or without decompensated cirrhosis (median S/CO value eries or transfusion as the main risk factors for HCV infection
41.2 versus 45.1, respectively; P = 0.28), and did not dif- had an obstetric/gynecologic indication for these procedures,
fer between women who were or were not recipients of may help to explain the higher frequency of women with HCV
transfusion (Table 4; Fig. 1A). Nevertheless, HCV RNA- infection, as compared with that of men [5,16]; however, this
positive patients had higher anti-HCV levels than women issue needs further investigation.
without detectable HCV RNA (Table 4; Fig. 1B), and this The levels of anti-HCV were higher in HCV RNA-
difference remained regardless the antecedent of transfusion positive than HCV RNA-negative women. Furthermore, the
(Fig. 1C and D). Anti-HCV levels directly correlated with the time elapsed from the acquisition of HCV to the diagnosis
elapsed time from transfusion to diagnosis of HCV infection correlated with the levels of anti-HCV antibodies, in women
(Fig. 2A), but not with age (Fig. 2B). After subgroup analysis, with transfusion as the putative route of infection. Hence,
this correlation was higher in HCV RNA-positive (Fig. 2C) as it is shown by the determination coefficient (r2 ) obtained
than in negative women (Fig. 2D). from the correlation of anti-HCV levels and duration of the
infection in HCV RNA-positive women, it is expected that
about 14% of the variation in anti-HCV levels is determined
4. Discussion by the state of chronicity. This is in agreement with a previous
study that analyzed 43 recovered and 34 chronically infected
In Mexico, the proportion of women with HCV infection women who were recipients of human Rhesus immunoglob-
might be higher [5,16] than that reported in other countries ulin contaminated with HCV genotype 1b [15]. This report
[30–32], as is observed in several populations [10,17,18]. We demonstrated that in women with chronic hepatitis C,
E. Chiquete et al. / Hepatology Research 36 (2006) 100–106 105

Fig. 2. Correlation between levels of anti-HCV antibodies (expressed as sample-to-cut-off [S/CO] ratio) and the elapsed time from the earliest transfusion to
the year of diagnosis (A), as well as age (B), in women who were recipients of blood products before 1993 (n = 50). Correlation between anti-HCV antibodies
levels and the elapsed time from the earliest transfusion to the year of diagnosis in the subgroup of HCV RNA-positive (C) (n = 34) and HCV RNA-negative
women (D) (n = 16). Regression lines (fit line, center) and the corresponding mean 95% confidence intervals (outlying lines) are depicted.

anti-HCV levels were higher whereas cellular and interferon vulnerability to oxidative stress [38] and the antecedent of
response were weaker than in patients with resolved infection transfusion before blood bank screening for HCV to a large
[15]. Thus, on one hand, there is a pattern favoring humoral inoculum that may imply a high number of viral genetic vari-
response in the chronic state; and on the other hand, a strong ants [39], than other sources of infection [40].
cellular immune response [15,33] and an initial low viral load In conclusion, the source of HCV infection influences the
[34] are associated with further resolved infection. It is possi- severity of the liver disease, and the duration of the infection
ble that a long duration of the infection enables a continuous affects the levels of anti-HCV antibodies, in transfusion-
reinforcement of the humoral response, thus affecting the associated hepatitis C. In most cases, surgery and transfusion
levels of anti-HCV antibodies. Nonetheless, certainly other had an obstetric or gynecologic indication, a finding that may
factors as the cellular immune competence, viral replication explain why more women than men are observed with HCV
reservoirs, and other complex characteristics inherent to infection in Mexico. Further studies are necessary for confir-
HCV may determine the rest of the variation of anti-HCV mation of these results.
levels. Our findings confirmed previous observations [15]
and add that, even in transfusion-associated hepatitis C, the
duration of HCV infection influences the levels of anti-HCV Acknowledgements
antibodies.
The frequency of occult HBV infection was lower than We thank to Dr. Jorge Segura, Dr. Miguel A Jiménez, Dr.
that reported in other studies [35,36], and HBV coinfection Guadalupe Becerra, Dr. Angeles Quintero and Dr. Benjamı́n
was not associated with the severity of the liver damage. The Cárdenas for their clinical support and kind attention given
clinical importance of occult HBV infection has been a very to this work. We are also indebted to Montserrat Maldonado
debated topic, with studies that report a significant correlation and Daniel Quezada for their friendship and assistance in
with the severity of the liver damage [14,37] and other stud- laboratory. CONACYT, Salud-2004-C01-025 to A.P.
ies reporting no association at all [35,36], even in populations
with a high prevalence of occult HBV infection [35]. Here,
independent predictors of cirrhosis at histologic level were
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