Hepatitis C virus genotypes in Cordoba, Argentina unexpected high prevalence of genotype 2
Re V.1, Lampe E.2, Fumiko Yoshida C.2, Mendes de Oliveira J.2, Lewis Ximenez L.2, Spinsanti L.1, Elbarcha O.3, Contigiani M.1
1Instituto
de Virología Dr. J. M. Vanella. Facultad de Ciencias Médicas, Universidad
Nacional de Córdoba;
2Fundação Oswaldo Cruz, Departamento de
Virologia, Laboratório de Hepatites Virais, Rio de Janeiro, Brazil;
3Cátedra
de Virología, Facultad de Ciencias Químicas, Universidad Católica de Córdoba
Abstract
To
determine hepatitis C virus (HCV) genotypes circulating in the central region
of Argentina, 96 consecutive anti-HCV positive subjects were studied. The
presence of HCV RNA was detected in 60 samples by RT-nested PCR of the 5’
noncoding region (5’ NCR). Genotyping was performed by restriction fragment
length polymorphism analysis of 5’ NCR region combined with PCR using
type-specific primers of the core region. The groups of individuals in this
study included hemophilia and hemodialysis patients, injecting drug users,
screened blood donors, and patients with acute or chronic liver disease, all
from Córdoba, Argentina. Overall, genotype 2 was the most prevalent (55.0%),
followed by genotypes 1 (38.3 %), and 3 (5.0%). Within genotype 1, subtype 1b
was the most prevalent. An unexpected high prevalence of genotype 2 (61.9%) was
found among patients with acute or chronic HCV infection (without known risk factors).
These figures differ from other cohorts from East-Argentina where genotype 1
has been found as the most prevalent. This indicates that regional differences
of genotype distribution might exist between Central and East Argentina.
Key words: Hepatitis C virus; HCV genotyping; HCV genotype 2.
Resumen
A fin de determinar los
genotipos del virus de la hepatitis C (HCV) circulantes en la región central de
Argentina, se estudiaron 96 individuos anti-HCV positivos. La presencia del ARN
de HCV se detectó en 60 muestras mediante RT-nested PCR de la región 5’
no codificante (5’ NCR). La genotipificación se realizó mediante restricción
enzimática y el análisis del polimorfismo de los fragmentos largos de la región
5’ NCR combinada con PCR usando primers tipo específico de la región del core. El grupo de individuos estudiados incluyó pacientes hemofílicos y
hemodializados, drogadictos intravenosos, donantes de sangre y pacientes con
enfermedad hepática aguda y crónica, todos provenientes de Córdoba, Argentina.
El genotipo 2 fue el más prevalente (55.0%), seguido por los genotipos 1 (38.3
%), con mayor prevalencia del subtipo 1b, y el genotipo 3 (55.0%). Una
inesperada alta prevalencia de genotipo 2 (61.9%) se encontró entre pacientes
con infección aguda o crónica por el virus HCV (sin factor de riesgo conocido).
Estos hallazgos difieren de otros resultados encontrados en trabajos realizados
con pacientes provenientes de la región este de Argentina, en los cuales el
genotipo 1 fue encontrado como el más prevalente. Esto indica que pueden
existir diferencias regionales en la distribución de genotipos HCV entre la
región centro y este de Argentina.
Palabras clave: Virus de la hepatitis C; Genotipificación de HCV; HCV genotipo 2.
Hepatitis C virus (HCV) infection, which is a major
cause of chronic liver disease, is strongly associated with progressive liver
pathology, including cirrhosis, hepatic failure and hepatocellular carcinoma.
Considering that approximately 3% of world’s population (170 million persons)
are infected, chronic hepatitis C remains a serious medical problem with
considerable burden on the different health care systems1.
HCV is an enveloped positive single-stranded RNA virus (9.6-kilobases)
classified under the Flaviviridae family, which has remarkable genetic
heterogeneity. Comparative analysis of complete or partial genomic sequences of
HCV from different geographic areas identified at least six major genotypes
(1-6) and a series of subtypes (a, b, c, etc)2. Although HCV
genotypes 1, 2, and 3 and their subtypes have a worldwide distribution, their
relative prevalence varies from one geographic area to another. In Europe and
in North and South America HCV subtypes 1a and 1b have been reported as the
most prevalent3, 4, 5. In Japan, subtype 1b is responsible for
most of the cases of HCV infection6. While HCV subtypes 2a and 2b
are relatively common in North America, Europe and Japan, subtype 2c is more
commonly found in Italy7. Genotype 4 has been found mainly in Central
Northern Africa (the principal genotype in Zaire and Egypt) and in the Middle
East8, and types 5 and 6 have been reported in South Africa and Hong
Kong, respectively9, 10.
The HCV genotype has been shown to have an important role in clinical
and histological features and on the response to antiviral treatment11, 12.
Genotype 1, in particular 1b, is a strong predictor for a poor response to
interferon (IFN) therapy13, 14, 15. Infection with subtype 1b seems
to result in a more severe liver disease leading to cirrhosis and possibly
hepatocellular carcinoma11,16,17. These features suggest that
subtype 1b isolates possibly represent more aggressive variants of the virus.
Improved responses to IFN are found in those patients infected with either HCV
genotype 2 or 3 when compared to patients infected with genotype 1 and 4. Thus,
HCV genotyping is relevant not only for molecular epidemiological studies but
is also potentially important in predicting the success of interferon
treatment. Furthermore, the sensitivity and specificity of serological and
virologic assays for detection of HCV may be influenced by the heterogeneity of
HCV.
These observations have led to an overall interest in determining HCV
genotypes. In Argentina, available data show that genotype 1, particularly
subtype1b, is the most predominant, but a considerable percentage of non-1
genotypes have also been detected. Genotypes 2 and 3 have been reported, as
well as genotypes 4 and 5 to a lesser extent18-24. For the region of
Córdoba, located in the center of the country, no data on HCV genotype are
available up to now.
The aim of the present study was to determine the genotypes of
circulating HCV isolates in the central region of Argentina, using RFLP of the
5’ NCR region combined with PCR type-specific primer of the core region.
Materials and Methods
Population: 96 consecutive anti-HCV
positive patients (47 male; mean age 40.7 yr-old, range 17-70 yr and 49 female;
mean age 53.4 yr-old, range 26-72 yr) were studied. All sera were collected
between January 2000 and September 2001, from subjects living in Córdoba,
Argentina, and referred to the Institute of Virology, School of
Medicine, National University of Córdoba. The samples comprised 49 patients
with sporadic hepatitis (47 chronic hepatitis and 2 acute hepatitis), 21
patients with parenteral risk of infection (9 dialysed, 7 hemophiliac, 3
intravenous drug users - IVDU - and 2 polytransfused) and 26 screened blood
donors. The presence of anti-HCV antibody was determined by ELISA test III kit
(Abbott, AXSYM system).
RNA
Extraction: Viral RNA was extracted from 100 µl
of serum with 750 µl of Trizol (Life Technologies, Rockville, MD), 1 µl
(10 µg) of yeast tRNA, and 200 µl of chloroform. The mixture was mixed by
pulse-vortexing for 2 min, incubated for 20 min at room temperature, and
centrifuged at 14.000
rpm for 20 min. Total RNA was precipitated by isopropanol and ethanol, air
dried, and then dissolved in 20 µl of diethyl pyrocarbonate treated water
containing 40U of recombinant ribonuclease inhibitor (RNAsin, Promega,
Madison, WI, USA).
Reverse
transcription (RT) and amplification by Nested PCR: RT- Nested PCR for 5´-non coding region was used to amplify a 280 nucleotide
fragment, as previously described25. First PCR was carried out with
30 cycles (denaturation at 94°C for 30 sec, annealing at 50°C for 90 sec, and
elongation at 72°C for 2 min). The second PCR was done, at the same PCR
conditions, with 25 cycles. PCR products were loaded onto a 1.5% agarose
electrophoresis gel and visualized by staining with ethidium bromide under
ultraviolet light. To avoid contamination, RNA extraction and reverse
transcription, pre-PCR reagent preparation, DNA amplification, and gel electrophoresis
of PCR products were performed in four separate rooms.
HCV
genotyping: HCV genotyping/subtyping was
deter-mined by two RT-PCR based assays, the restriction length polymorphism
analysis (RFLP) of the 5´noncoding region (5´NCR), and a nested PCR with type
specific primers following primary RT-PCR with consensus primers designed for
the core region of HCV genome26.
RFLP: HCV genotyping by RFLP was carried out using the restriction enzymes AvaII
and RsaI on PCR-amplified from 5’ NCR as previously described27.
Briefly, 10 µl of nested PCR products were digested separately with 5 units of
restriction enzymes Rsa I and Ava II (Life Technologies).
After incubation for 1.5 hs, restriction fragments were visualized on 3%
agarose gel electrophoresis stained with ethidium bromide. This RFLP assay
allows to distinguish between the genotypes 1, 2, 3.
Type-specific
PCR assay: This was accomplished by means of the nested PCR
assay originally developed by Okamoto and co-workers28 for subtyping
of genotypes 1 and 2, and modified by Widell29 to identify genotype
3. All samples positive for the 5’ NCR were analysed. Sequences of the HCV core
region were detected after synthesis of cDNA and nested PCR with conserved
primers covering positions 439 to 751 of the HCV genome26. Both,
first and second PCR, were done with 35 cycles (94°C for 45 sec, 55°C for 1
min, and 72°C for 1 min 15 sec), allowing the synthesis of 313 base pair (bp)
amplicons. Viral sequences amplified from the first PCR reaction with conserved
primers of the core region were subjected to a second round of PCR with
respective type-specific primers in two separate reactions, one for the
amplification of genotypes 1a, 1b, 2a, 2b with primers type-specific for these
genotypes and the other for the amplification of genotypes 1b, and 3, according
to the established protocol29. Twenty five cycles of denaturation
(94°C for 60 sec), annealing (63°C for 60 sec) and extention (72°C for 90 sec)
were undertaken.
Statistical
analysis: Ages are reported as mean (standard deviation) and
were compared between the genotype groups using Student’s t-test. Chi-square
test was used to evaluate gender distribution using STATA 6 (Stata Corporation,
College Station, Texas). Statistical significance was established at P < 0.05.
Results
Among 96 patients anti-HCV positive, 60 (62.5%) were HCV RNA positive by
PCR for 5’ NCR. The presence of HCV RNA was detected among 42/49 (85.7%) acute
or chronic hepatitis patients, in 11/21 (52.4%) of risk group and 7/26 (26.9 %)
blood donors. Females tested positive more frequently than males (65.3% vs.
59.6%; P < 0.05).
The 5´ NCR was chosen for quick typing by RFLP analysis of 280 bp nested
PCR product using the restriction enzymes AvaII and RsaI. By
using both enzymes simultaneously it was possible to get initially information
about genotypes 1, 2 and 3. In this way, type 1 was identified in 23/60
(38.3%), type 2 in 33/60 (55.0%), type 3 in 3/60 (5.0%) of the samples and one
(1.7%) patient gave a indeterminate pattern.
Genotyping by using type-specific primers of the core region requires an
initial amplification by PCR of genomic sequences with conserved primers
followed by subtype-specific primers. Seven samples unfortunately did not have
enough serum for PCR testing in core region thus, fifty three samples were
submitted to PCR type-specific assay. Genotyping was successful in 39 of them.
In 14 samples the HCV genotype could not be identified (all type 2 by RFLP
assay), because of unsuccessful PCR amplification with the type-specific
primer, and one sample gave indeterminate pattern of bands. Therefore, the
genotype results among the 39 patients by PCR type-specific assay showed
type 1a in 5 (12.8%), type 1b in 16 (41.0%), type 2b in 16 (41.0%), type 3 in 1
(2.6%) and indeterminate in 1 (2.6%) of patients.
The genotype distribution by RFLP or by PCR type-specific assay
in the all (n=60) positive HCV RNA samples showed that 55% were identified as
type 2, 38.3% as type 1, 5% as type 3, and one sample (1.7%) gave indeterminate
results by both assays. Within genotype 1, subtype 1b was the most prevalent.
None of the individuals was infected with more than one genotype. Table 1 shows the genotype distribution according to age and
gender. A predominance (68.7%) of genotype 2 was observed in the female gender.
However, this finding was not statistically significant (p=0.059). The mean age
of patients infected with genotype 2 was slightly higher, but no significant
association was observed between age and genotype.
In the patients with chronic or acute hepatitis, genotype 2 was more prevalent with 26 (61.9%) out of 42 patients infected, genotype 1 accounted for 33.3% (n=14) and genotype 3 for the remaining 4.8% (n= 2) (Table 2). In our group of patients with risk factor for HCV infection (hemophiliac, hemodialyzed and IVDU; n=11) the presence of genotype 1 was identified in 5 (45.4%), genotype 2 in 4 (36.4%) and genotype 3 in 1 (9.1%) patient. One hemophiliac patient was indeterminate. Among the 7 blood donors, only genotype 1 (n=4) and 2 (n=3) were detected.
Discussion
This study shows that in the area of central Argentina, genotype 2
accounts for most of the cases of HCV infection. The genotype distribution in
our population showed that 55% were infected with HCV type 2, 38% with type 1,
and 5% with type 3. This high prevalence of type 2 among subjects living in
Córdoba is surprising and, to our knowledge, this is the first study of the
genotype distribution in this area.
These figures differ from other cohorts from Argentine patients in whom
genotype 1 has been found as the most prevalent, followed by genotype 2 and 318,
19, 21, 22, 24. Quarleri et al.20 detected genotype 1
in 70.7%, genotype 2 in 21.9% and genotype 3 in 7.3% in different Argentine
groups. More recently, Alfonso et al.23, reported genotype 1
in 61.4%, genotype 2 in 12.3%, genotype 3 in 23.7% and genotype 4 in 2.6% of
HCV isolates from 114 infected patients. Despite the presence of genotype 2
being detected with relative high frequency in these different studies, in none
of them prevalence as high as 55% has been found, as we have in the present
study. However, those previous studies were probably performed on patients from
the City of Buenos Aires region. This discrepancy indicates that regional
differences of genotype distribution might exist between Central and East
Argentina. Substantial differences in HCV genotype distribution between close
geographical regions had also been observed in Taiwan. These findings implied
that independent HCV outbreaks with introduction of particular HCV isolates
could lead to different HCV molecular epidemiology in distinct areas and
resulting in the diverse HCV genotype distribution30.
The genotype distribution of HCV infection found in our study population
suggests a pattern similar to that reported in Italy, where most
community-acquired cases studied so far belong to genotype 2. In a study
carried out in the population of a town in Southern Italy, the HCV genotype
distribution shows type 1b in 50.7%, type 2b in 0.7%, type 2c in 44.6%, type 3a
in 2.7%, and type 4 in 1.3%31. Ravaggi, et al.32,
also reported 54.2% (39/72 ) of genotype 2 prevalence, especially subtype 2c,
in Italian patients. Similar HCV genotype distribution (around 50% of genotype
2) has been observed in other cohorts from both Southern and Northern Italy7,
33, 34.
The fact that type 2 was detected in 61.9% of patients with acute or
chronic HCV infection without known risk factor highly suggests that this type
may account for most of community acquired HCV in Central Argentina. Patients
with risk factors for HCV infection enrolled in the present study were very few
and therefore we are not allowed to reach any conclusion of genotype
distribution within this group. The distribution of the two most common HCV
viral types (2 and 1) was not statistically different in terms of mean age or
gender and suggests that they may have had a simultaneous spread in this
community.
One of the most widely used methods for genotyping HCV is a polymerase
chain reaction (PCR) based on amplification of specific sequences in the core
region by subtype-specific primers, as described by Widell et al.29.
However, this assay, which recognizes the five most common HCV subtypes (1a,
1b, 2a, 2b and 3), was developed before the identification of new genotypes and
may be inadequate to be used in some geographic areas.
Major problems were found in the identification of isolates belonging to
type 2, since no genotype determination was achieved in 14 samples identified
as genotype 2 by RFLP assay, because of unsuccessful PCR amplification with the
type-specific primer of the core region. In the type-specific methods used,
only primers for the most common subtypes, 2a and 2b, are employed, so that
probably our samples could belong to another 2 subtype and could not be
amplified. In fact, sequence analysis in the core region from 4 of these
samples demonstrated that they belong to subtype 2c (unpublished author’s
data). This could explain the difference in sensitivities of the two genotyping
tests used. Sequences studies on more samples are ongoing in order to better
define the subtype 2 circulating in our region. The presence of subtype 2c in
Argentina has been related by Quarleri et al.20, by sequence
analysis in the core and NS5 regions from a sample showing discrepancies using
two different methodologies; moreover, they found a strong predominance of
subtype 2a/c (16/18) over 2b subtype by sequence analysis in the 5’ NCR region.
For detecting subtypes 2c, reported to be circulating in Italy, Spada et al.7
propose to adapt the PCR method for genotyping including primers specific to
these genotypes. Taking into account the identification of subtype 2c by
sequencing analysis, and recalling the great difficulty we had to identify
genotype 2, it seems plausible that at least for our local strains of HCV, it
might be required to use core-specific primers to subtype 2c, for correct
genotype classification. Further investigation with more sequencing samples is
needed to obtain the evidence that the genotype 2c isolates observed in this
study are significantly different from other isolates previously characterised
by sequence analysis.
In conclusion, the results reported here in point to the high prevalence
of HCV genotypes 2 in patients living in the Central Region of Argentina, and
most importantly that these findings have consequential therapeutic
implications in view that infections with HCV 2 or 3 were independent predictors
for sustained response to IFN monotherapy15,35 or IFN/ribavirin
combination11,36. In general, patients with the genotype 1 infection
should receive 48 weeks of therapy, and those with genotypes 2 or 3 infection
only 24 weeks. Viral load estimations are problematic because of normal
fluctuations (up to 0.5-10 log), assay variability and lack of a universally
accepted standard; thus, viral load testing is not recommended routinely at
present37. Furthermore, the determination of HCV genotypes present in
different regions of Argentina is very important for epidemiological
surveillance, blood-donor screening and adequacy of foreign serological and
genotyping tests.
Acknowledgements: This study was supported in part by grants from CAPES-cordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil), and SeTCIP-Secretaría para la Tecnología, la Ciencia y la Innovación Productiva (Argentina).
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Recibido:
26 de diciembre de 2002
Aceptado:
21 de abril de 2003