DISTRIBUTION OF HEPATITIS C VIRUS GENOTYPES IN PATIENTS
ON REGULAR HEMODIALYSIS PROGRAM
1
Running head: HEPATITIS C VIRUS GENOTYPES IN HEMODIALYSIS PATIENTS
KEY WORDS: Hepatitis C virus, anti HCV, HCV genotypes, reverse transcription polymerase
chain reaction, chronic HD patients.
Authors: Jasna Slaviček1, Zvonimir Puretić1, Smilja Kalenić2, Božica Rebrović2, Blaženka
Grahovac3, Snežana Glavaš-Boras1, Zvonimir Mareković1, Vladimir Slaviček4 , T.Golubić,5
Institution:
1
Urology Department, University Hospital Centre Zagreb, Rebro, Kišpatićeva 12, 10000 Zagreb
2
Clinical Laboratory Diagnosis Institute, Microbiology Laboratory, University Hospital Centre
Zagreb, Rebro, Kišpatićeva 12, 10000 Zagreb3 Transfusion Medicine Institute, Petrova 3, 10000
Zagreb ,4Traumatology Hospital, Draškovićeva 19, 10000 Zagreb , 5 Department of Infectology
Hospital Čakovec
Address of author: Jasna Slaviček M.D. M.S.
Dialysis center, Urology Department
University Hospital Center
Zagreb Kišpatićeva 12, 10 000 Zagreb
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J. Slaviček
Abbreviations
HCV
hepatitis C virus
HD
hemodialysis
HCV RNA
hepatitis C virus RNA
RT PCR
reverse transcription polymerase chain reaction
EIA
enzyme immunoassay
anti HCV
hepatitis C antibodies
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J. Slaviček
ABSTRACT
Background and purpose. Hepatitis C (HC) prevalence is relatively high in patients on regular
hemodialysis (HD) program. Variations in the viral genome may lead to different clinical
manifestations of the disease and to variations in the response to treatment. The distribution of
HCV genotypes is related to the geographical area and depends on population-specific risk
factors. The purpose of the study was to assess the distribution of HCV genotypes in patients on
regular hemodialysis.
Matherial and methods. Testing for HCV RNA was performed in 95 patients on regular
hemodialysis in Dialysis Centre, Urology Department, University Hospital Center Zagreb, using
Amplicor HCV test and quantitative Amplicor Monitor test. Inno Lipa HCV test was used for the
determination of HCV genotype.
Results: HCV RNA was positive in 40/95 patients (53.6%). In the group of 56 anti HCV+ (EIA
3 test), HCV RNA was positive in 38 (67.6%)patients. Positive HCV RNA was found in 2 anti
HCV negative patients who later developed clinical signs of acute hepatitis C. Genotype 1b was
dominant (29/40, 72.5%), followed by type 3 (8/40, 20%). Types 4 and 1a were found in two
patients. In one patient HCV genotype could not be determined. All HCV RNA positive patients
presented with viral load ranging from 5x103 to 6x106 copies/ml.
Conclusion. The distribution and frequency of the HCV genotypes in hemodialysed patients
followed the general distribution pattern recorded in Croatian blood donors with a predominance
of types 1b and 3a that were found in 71.6% and in 10,7 % of cases respectively. The presence
of four different HCV genotypes may be explained by the relatively high patient turnover in the
4
Center, where patients from other centers come for the treatment of complications of vascular
access.
J.Slaviček
INTRODUCTION
Hepatitis C virus (HCV) was discovered 1989 as the etiologic agent of non-A non- B hepatitis
( 1). It is transmitted primarily by the pareneteral route, and the infection tends to became
persistent in approximately 60-80 % of infected persons. It can cause a wide spectrum of liver
disease , ranging from asymptomatic acute hepatitis to cirrhosis and hepatocelular carcinoma
(2).Prevalence of antibody to HCV ( anti HCV) positivity among chronic hemodialysis( HD)
patients averages 10 % with some centers reported rates greater than 60 %(3). Both incidence
and prevalence studies have documented an association between anti HCV positivity and
increasing years on dialysis , independent of blood transfusion (4). These studies as well as
investigation on dialysis –associated outbreaks of hepatitis C , indicate that HCV transmission
might occur among patients in a HD center because of incorrect implementation of infection
control practices (5,6). Patients on regular HD are a patient group at a high risk for the
development of hepatitis C which is an important agent of liver disease in this clinical
setting.The disease prevalence varies from 2% to 62% . In Northern Europe, for instance, this
prevalence is below 10%, while in Eastern Europe is higher than 40% (7,8).In the past the use of
non screened blood and blood products was a major mode of transmission of HC in HD centers
At present , posttransfusional hepatitis C occurs in less than 5 % of hemodialysis patients.
Nosocomial infection is considered the major mode of transmission of HC in HD centers (9-10).
Exact mode of transmission of HCV in the hemodialysis population remain unresolved.
5
Hepatitis C virus is a heterogenous virus by nature of its replicative strategy. Major differences in
the nucleotide sequences of HCV isolates are found throughout the genome and indicate the
J. Slaviček
presence of distinct HCV genotypes, defined as having nucleotide divergence of more than 20
%. The wordwilde distribution of the genotypes is variable : HCV genotypes 1,2 and subtype 3a
appear to be the most prevalent in North America, Europe and Japan , and genotype 4 is
common in Africa.Among American patients with chronic hepatitis C , nearly 75% have
genotype 1 ( 11). Recent data for Croatian patients revealed the genotype 1b with 49 % as most
common in patients with chronic hepatitis C.(12). HCV can be classified into six major genotypes
according to nucleotide sequence in different regions of the genome.( 13,14) Genotypic
differences have been observed with regard to the viral load , severity of liver disease and
response to interferon therapy.( 15,16). The relative prevalence of HCV genotypes shows
marked geografical variations with obvious clinical implications. Studies of HCV genotypes in
patients on hospital hemodialysis are still insufficient. No study on HCV genotypes distribution
in regular HD patients has been published in Croatia so far.
The aim of our study was to determine the HCV genotype distribution in our patients on regular
HD.
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J. Slaviček
PATIENTS AND METHODS
Ninety-five (57 male and 38 female) patients on regular HD in the Dialysis Center, Urology
Department, UHC Zagreb, were included in the study. Only patients regularly dialyzed in the
Center were included. The patients are dialyzed three times weekly in three shifts. As a rule,
patients are always dialyzed on the same machines, except when the machine is out of order; then
it is replaced with a spare one. HCV positive patients are dialyzed on separate machines, in the
same room with HCV negative patients, but in their half of the room. Hepatitis B positive patients
are dialyzed in a separate room. A nurse normally cares for 4 HD patients. The staff caring for
HCV positive patients usually does not attend to HCV negative ones, except if there are problems
with AV fistula puncture. All dialysis equipment is disposable. Filters are not reused. Dialysis
machines are sterilized with hypochlorite and citric acid after each shift. Hepatitis markers,
HBsAg and anti HCV are routinely assessed trimesterally, and anti HIV twice yearly in patients
and twice yearly in the personnel. Liver biochemistry analyses (AST, ALT, alkaline phosphatase,
gamma-glutamyl transpeptidase (-GT), bilirubin) are routinely performed monthly.
An earlier testing confirmed an anti HCV positivity in 54/95 HD patients (EIA 3rd generation,
Ortho, Abbott and RIBA 3, Ortho).
Serum HCV RNA was determined by means of PCR method ( Amplicor HCV PCR kit, Roche
Systems, Basel , Switzerland), where reverse transcription (RT), amplification reaction and nonisotope hybridization are used. The sera were stored at 4-8o C if the test was performed within 72
hours, or at -20 o C or -80 o C if the test was done after a longer period of time. HCV genotypes
were assessed using reverse hybridization reaction, the so-called line probe assay (LiPa,
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J. Slaviček
Innogenetics, Zwijndrecht, Belgium). In all patients serum aspartate (AST), alanine
aminotransferase (ALT) were measured spectrophotometrically using multi-enzyme
autoanalyzer. Here are the Hospital laboratory normal values: 6-26 U/L for AST and 6-34 U/L
for ALT.
RESULTS
Demografic features of our patients are shown in table 1.
In 40/95 ( 53.6%) patients on regular HD tested, HCV RNA was found positive.
In the group of 56 anti HCV positive patients, HCV RNA was positive in 38 patients (67.6%).
(Table 2). A positive HCV RNA was also found in 2 anti HCV negative patients. In the later
course of the disease, they both developed acute hepatitis C with elevated transaminase levels and
a seroconversion after 6-8 weeks. In one patient with a clinical presentation of acute hepatitis C,
who became also anti HCV positive within a month, in spite of repeated testing in two different
laboratories and using two different techniques, routine and in house method, serum HCV RNA
was repeatedly negative.
Viral load varied between 5x103 and 6x10 6 copies per milliliter of serum. HCV RNA positive
finding correlated well with transaminase elevation ( Table 3).
We did not find any significant correlation between viral load and transaminase levels. The
patient with the greatest load (6x106) had quite normal transaminase levels for many years.
This can be explained by an extrahepatic proliferation of the virus. The virus genotype assessment
proved that type 1b is predominant in our Center. It was found in 29/40 patients (72.5 %), and
type 3 in 8/40 ( 20%), respectively. The type 1a was confirmed in one patient and
in one
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female patient type 4, which is otherwise exceptionally rare (Table 4). Due to a very low number
of copies, genotype could not be assessed in one patient.
DISCUSSION
HCV infection is high prevalent in HD patients (4). HCV RNA was found in 67.6% of anti
HCV positive patients. Among EIA 3 positive samples which were negative to RT-PCR method,
32.4% of patients either recovered from the HCV infection or their viral load is very low, below
the sensitivity limit of RT-PCR, or they suffer from a fluctuant viremia . In a later testing we
confirmed that HCV RNA became positive in two anti HCV positive patients, who were HCV
RNA negative in the first assessment. Finally, HCV RNA negativity obtained by using RT-PCR
technique in HCV infected patients may be caused by an exclusive localization of HCV in the
liver or peripheral blood mononuclear cells ( PBMC) or in other non-blood compartments (16).
This was the case with the patient in whom transaminase levels abruptly increased (ALT 340
U/L) and who seroconverted after 6 weeks, but remained HCV RNA negative over the whole
follow-up period of 12 months. In 2 patients a positive HCV RNA was the first sign of HCV
infection. In these patients transaminase levels increased after a week or two, and over 6-8 weeks
the patients became anti HCV positive. HCV viremia with very high blood levels of virus
particles, without biochemical signs of liver inflammation and with a negative HCV , could
reflect extrahepatic proliferation of the virus (15, 16). This was the case with our patient with the
greatest load (>6x106 copies/ml), but with normal transaminase levels ( ALT 6-8 U/L, AST 4-6
U/L, GT 10 U/L) over the entire 2-year follow-up period.
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J. Slaviček
There is a relatively small group of patients with a detectable serum HCV RNA, but who were
still anti HCV negative. (17,18). We found HCV RNA in the serum of 3 anti HCV negative
patients
At the repeated testing at the same laboratory and additional controls in other institutions,
however, all three patients proved HCV RNA negative. We explained this as a contamination.
HCV has a high mutation rate. It exists in nature as a population of diverse, but closely related
genomes (11,13,21).
The determination of HCV genotypes is based on the comparison of nucleotide sequences of the
virus genome and confirmed by phyllogenetic analysis. Both blood donors and patients with
chronic hepatitis from countries in Western Europe and the USA all show frequent infection
with genotypes 1a, 1b,2a,2b,and 3a, although the frequencies of each may vary. There is a trend
for relatively more frequent infection with type 1 b in southern and eastern Europe. Type 1a and
3 a are more common in intravenous drug users in Europe (7,11,20,22).In Croatia a study of HC
genotypes was done in blood donors.(12). The study proved that type 1b is dominant in Croatian
blood donors, then types 1a and 3. Type 4 is very rarely found. In our HD patients type 1b is
prevalent, similarly to HD populations in Korea, France and Japan .
Type 3 is less frequent. Type 4 was found in only one female patient. This type is very rare in
Europe and has been described in central Asia and in the Middle East (11).
It was detected in a lady who was previously submitted to a surgery for the correction of a
wrongly healed shin bone fracture. During the surgery, the patient received a blood transfusion, so
10
it may have been a posttransfusion hepatitis. The high prevalence of HCV infection in
hemodialysis patients has been attributed not only to the frequency of blood transfusion among
J.Slaviček
these patients but also to increasing years on dialysis , suggesting that HCV may be transmitted
between patients in the dialysis unit probably as a result of poor infection control practices ( 4,9).
A precise route of transmission is not known. A transmission by contaminated hands from
medical personnel or from patient to patient is presumed (4,6,14,20).
The introduction of erythropoietin in the treatment of renal anemia and obligatory screening of
blood donors to anti HCV has significantly reduced the spreading of posttransfusion HC. It is
estimated that the risk for this type of hepatitis is 1: 100 000 blood units (6,10, 18).
In smaller HD centers one HCV genotype only was found, which confirms the hypothesis of
nosocomial spreading of HCV within a center.(12,17)
Several different HCV genotypes in the investigated material can be explained by a relatively
high patient fluctuation in our Center, to which patients from other smaller centers are referred for
the treatment of vascular access complications. Spreading of HC in our Center is probably also a
result of nosocomial infection, as a number of HCV positive patients have never received a blood
or blood derivatives transfusion. There have been no data about other possible sources of
infection, e.g. drug abuse or tattooing either.
We would like to stress the need for a strict observing of measures of infection prevention in HD
centers.
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J. Slaviček
Table 1
Demografic features of our hemodialysis patients
N
95
Gender
57 m
Age
16 yrs –75 yrs
HD duration
1,4 yrs – 18,5 yrs
,
38f
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Table 2 Relation between HCVRNA positivity and anti HCV positivity.
Anti HCV+ (EIA 3)
Anti HCV – ( EIA 3)
n
%
n
%
HCV RNA +
40
71.5
2
5,1
HCV RNA -
16
28.5
37
92,3
Total
56
100
39
100
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Table 3
Correlation between transaminase values and HCV RNA
HCV RNA +
HCV RNA -
ALT elevated
21
5
ALT normal
19
50
Total
40
55
p <0,5
Legend : ALT normal ( 6-26 U/L)
ALT elevation ( exceeding the upper normal reference level)
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Table 4
Distribution of HCV genotypes in hemodialysis patients.
HCV genotype
N
%
1b
29
72,5
3
8
20.
1a
1
2,5
4
1
2,5
unknown
1
2,5
Total
40
100
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