Influence of Hepatitis B Virus (HBV) Genotype on the Clinical Course of Disease in Patients Coinfected with HBV and Hepatitis Delta Virus
0
Retrovirology Laboratory, Federal University of Sao Paulo
,
Sao Paulo
,
Brazil
1
Viral Hepatitis Outpatient Center, Federal University of Amazonas
,
Manaus
2
Laboratory of Research, Amazonas Blood Center
Objective. We evaluated the influence of hepatitis B virus (HBV) genotype on the course of disease in patients coinfected with HBV and hepatitis delta virus (HDV). Methods. We evaluated HBV genotypes in 190 patients, 140 of whom had chronic HBV monoinfection and 50 of whom had chronic HBV-HDV coinfection. Real-time polymerase chain reactions for the amplification of HBV DNA and HDV RNA were developed, and we compared the patient groups with respect to HBV genotype, viral load, alanine aminotransferase (ALT) and bilirubin levels, and disease severity. Results. Coinfected patients had higher ALT and bilirubin levels as well as a higher prevalence of liver cirrhosis and liver carcinoma. ALT levels were higher among individuals coinfected with HDV and HBV genotype F than among individuals infected only with HBV genotype F. Among HDV-HBV- coinfected patients, HDV load was lower among those infected with HBV genotype A than among those infected with HBV genotype D or genotype F. Conclusion. Liver inflammation and HDV load are influenced by HBV genotype in individuals coinfected with HBV and HDV. Hepatitis B virus (HBV) genotypes A, D, and F have been found in the Amazon basin, and genotype F is the most prevalent among Amerindian populations [1, 2]. Studies have shown that
-
coinfection with HBV F and hepatitis delta virus (HDV)
genotype III has played a role in increasing the severity of the clinical
manifestations of hepatitis found in this region [3, 4]. However,
few studies have attempted to clarify the role of HBV genotypes
in the clinical evolution of HBV-HDV coinfection [5]. In the
present study, we analyzed and compared patients with HBV
monoinfection and patients with HBV-HDV coinfection,
evaluating viral genotypes, viral load, serologic results, and clinical
characteristics.
Methods. From February 2003 through November 2006, we
studied 190 patients with chronic HBV infection. We excluded
patients who were coinfected with hepatitis C virus, human T
cell lymphotropic virus, or human immunodeficiency virus. We
also excluded patients who were receiving treatment with
interferon or antiviral medication, patients with alcoholism, and
patients who were users of illegal drugs. Participants were from the
city of Manaus in the State of Amazonas, in northern Brazil. This
study received institutional review board approval, and all
participants provided written informed consent.
Of 190 participants, 140 (104 men and 36 women) had HBV
monoinfection and 50 (32 men and 18 women) had HBV-HDV
coinfection (defined as 2 consecutive serological tests positive
for antibodies to HDV and HBV surface antigens). The time
since diagnosis was defined as the interval in months between
diagnosis of the infection and inclusion in the study. Subjects
were divided into 4 diagnosis groups, defined as follows: (1)
patients with asymptomatic carriage (AC), who had normal
hematological, biochemical, and ultrasonographic parameters; (2)
patients with chronic hepatitis with evidence of liver
inflammation (CH), who had had elevated alanine aminotransferase
(ALT) levels for 6 months, without evidence of liver cirrhosis;
(3) patients with liver cirrhosis (LC), who had esophageal
varices, splenomegaly, and thrombocytopenia or imaging findings
consistent with the same; and (4) patients with hepatocellular
carcinoma (HCC), as identified by computed tomography or
magnetic resonance imaging.
For HBV genotyping, DNA was amplified with primers to the S
gene. Subsequently, internal primers were employed to identify
genotypes AH [6 8]. HBV quantification was performed with
realtime quantitative reverse-transcriptase polymerase chain reaction
(qRT-PCR). The primers used (pre-S/S region) were
5'-CAACCTCCAATCACTCACCAA3' and
5'-ATATGATAAAACGCCGCAGACAC-3', and the probe was
5'-FAM-TCCTCCAATTTGTCCTGGTTATCGCT-3'-TAMRA.
For the HBV internal control, a partial sequence of the HBV
genome was internally modified. After amplification and
cloning, the insert was cultivated and purified. For detection of the
internal control, the probe incorporated the fluorochromes
HEX and TAMRA. A positive control for HBV was prepared
using the sequence to be quantified in the reaction, the PCR
product of which was cloned and cultivated. After purification
and determination of the concentration of the clone, serial
dilutions were standardized, in international units per milliliter, by
use of the OptiQuant HBV DNA Quantification Panel.
qRT-PCR was used for quantitative detection of HDV RNA.
The target region corresponded to nt 9421032 [9], and the
probe was
5'-FAM-CACCCTGGGACCCAGTAATACCCGG3-TAMRA.
For the HDV internal control, a partial HDV sequence was
internally modified and amplified by use of qRT-PCR to
produce synthetic DNA, and the product was subcloned. For
detect (...truncated)