Description of Hepatitis B Virus Genotypes in Selected Groups of Subjects from Paraguay and Brazil

Hepatitis B virus genotyping was conducted among 11 560 healthy blood donors and haemodialysis patients in Brazil and Paraguay. Geno types A, D and F were found in Brazil and genotypes D and F in Paraguay. This is the first report showing HBV genotype diver sity in individuals in Paraguay.


INTRODUCTION
Hepatitis B Virus Infection (HBV) is a relevant public health problem, with 2 billion people infected worldwide [1] . Effective vaccines against HBV are available starting from 1982 [1] , however not all countries adopted those recommendations in an official way.
An important epidemiological aspect of HBV is the tendency to establish closed epidemiological cycles, hence, detailed knowledge is essential to design and improve specific prevention and control strategies [2] . However, this notion is still incomplete since the number of isolates analyzed in some parts of the world, including South America, is small [3,4] .
HBV strains are classified into eight main genomic groups, designated A-H [5,6] showing a global distribution. In Brazil the most frequent genotypes were A, D and F [7] .
The objective of this study was to detect and characterize HBV strains circulating among selected populations in Brazil and Paraguay, including healthy blood donors and haemodialysis patients. HBV viral diversity in Paraguay is evaluated here for the first time.

MATERIALS AND METHODS
A total of 11560 serum samples from healthy blood donors and haemodialysis patients in Brazil and Paraguay were divided into four groups. Characteristics and provenance of studied population groups are shown (Table 1).
Samples were tested for HBsAg by Enzyme immunoassay (MONOLISA® HBsAg PLUS, BIO-RAD) and "indeterminate" samples were also registered. The haemodialysis groups were also tested for anti-HBc by Enzyme immunoassay (MONOLISA® Anti HBc PLUS, BIO-RAD). Nucleic acid was extracted from serum as previously described [8] . Detection of HBV DNA and genotyping was carried out in the HBsAg positive samples, in the "indeterminate" samples and the HBsAg-/ anti HBc+ samples; the methodology used was nested PCR using type-specific primers for each genotype, originally reported by Naito et al. [9] with slight modifications. We amplified the HBV genome using the universal primers followed by two different amplifications containing type-specific inner primers designed on the basis of the differences in size of the genotype-specific bands [9] . PCR was carried out in a tube containing 50 µl of a reaction mix with the following components: 50 ng of each outer primer, 200 mm of dNTPs, 1 U of Taq   , type D to F specific, antisense) An "M" represent a nucleotide that could be either an A or a C: a "Y" represents a nucleotide that could be either a C or a T. nt, nucleotide. From Naito et al. [9] DNA polymerase (Life Technologies) and 1x PCR buffer containing 1.5 mm MgCl2 and 5 µL target DNA. The thermocycler. Cycling parameters were: initial incubation for 5 min at 95°C, followed by 40 cycles consisting of 94°C for 20 s, 55°C for 20 s and 72°C for 1 min. Two second-round PCRs were performed for each sample, termed mix A and mix B reactions. Mix A consisted of a PCR mix able to detect genotypes A to C and mix B D to F. A table with universal and genotype specific second-round primers is shown ( Table 2). 1-µL of the first PCR product was added to two tubes containing the second sets of each of the inner primer pairs. Reactions proceed as described for first-round amplifications. HBV genotypes were determined by genotype-specific DNA band sizes in a 3% agarose gel.

RESULTS AND DISCUSSION
HBsAg was detected in 54 of 11560 individuals. Among Paraguayans samples, 12 (8.3%) belonged to the haemodialysis unit and 6 (0.2%) to the blood transfusion service. In Brazil, 3 patients (3.8%) belonged to the haemodialysis group and 33 (0.4%) to the blood transfusion service (Table 1). 4(0.03%) of all samples were considered "indeterminate". We found an anti-HBc prevalence of 22.7% in Asunción and of 12.8% in São Paulo.
HBV DNA was amplified in 61 samples, 54 HBsAg positive, 4 "indeterminate" and 3 with a HBsAg-/anti-HBc+ profile; 1 from Asunción and 2 from São Paulo. HBV genotyping was accomplished in all the 61 HBV DNA positive samples. As shown in Fig. 1, type-specific PCR products were recognized by their distinct sizes in gel electrophoresis. Fig. 1: The typical electrophoresis patterns of PCR products from different HBV genotypes found as determined by PCR genotyping system.. M, molecular size standards Table 3: Genotypic distribution of HBV among different groups, determined by PCR using type-specific primers Group, determined by PCR using type-specific Genotypes D and F were found in Asunción, genotype D was found in 13 (93%) of the Paraguayan haemodialysis patients. Genotypes A, D and F were found in São Paulo (Table 3). We found that 0.4 and 0.2% of the samples stored in blood banks, in the cities of São Paulo and Asunción, respectively, are HBsAg positive. These percentages fall within the expected range (between 0.3% and 13%) for HBV infections in different regions in Brazil [10] . Prevalence in blood bank samples was lower than that observed in individuals submitted to haemodialysis, being 8.3% in Asunción and 3.8% in São Paulo, probably due to the acquisition of HBV during the procedure.
From 223 studied patients belonging to the two haemodialysis groups, 12.1% were negative for HBsAg and positive for anti-HBc. A recent study showed that at least 35% of patients with hepatocellular carcinoma were HBsAg negative but harbored HBV DNA in the liver [11] .
A study about HBV variant distribution done in haemodialysis units in the cities of Rio de Janeiro and São Paulo demonstrated that genotypes A and D are prevalent in Rio de Janeiro, while only genotype D was detected in São Paulo [12] . We found genotypes A, D and F in São Paulo. The most prevalent genotype in this city was A followed by D and F. In Paraguay, we detected the genotypes D and F on the Blood Bank and genotype D in the haemodialysis units. In this case, the result may reflect an mechanism of infection in patients undergoing the procedure. Additional studies done in the general population would clarify if genotype D in Paraguay is linked to haemodialysis. More detailed studies using DNA sequence data will help to evaluate these questions. Since this is the first study showing the HBV genotype distribution in Paraguay, we believe that this study may contribute to the understanding of the HBV epidemiology in Paraguay in those groups.