Occult Hepatitis B Infection among Hemodialysis in Tabriz, Northwest of Iran: Prevalence and Mutations within the S Region

Abstract

Regardless of the extensive screening for the detection of hepatitis B surface antigen (HBsAg), hemodialysis (HD) patients are still severely at the risk of occult hepatitis B virus infection (OBI), especially in developing countries. OBI is defined as the presence of HBV DNA with undetectable HBsAg in the liver and/or Serum. This study aims to determine the prevalence of OBI in HD patients in Tabriz Province, northwest of Iran, and inquire about the mutations in the detected HBsAg. In this cross-sectional descriptive study, ELISA method assessed serum and plasma samples of 118 HBsAg-negative patients undergoing HD treatment for HBV serological markers (HBsAg and Anti-HBc). Specific primers by nested polymerase chain reaction have been utilized to examine HBV DNA; also, direct sequencing of surface genes was carried out to characterize the viral genotypes and S gene mutations. Finally, followed by real-time PCR, the quantity of viral load in OBI-positive patients was determined. A total of 118 HD patients were included (63.6% were male and 36.4% female), with an overall mean age of 60.8 ± 12.8 years old. The prevalence of antihepatitis B core antibody (Anti-HBc) in the study population was 26.3% (31/118). Five patients (4.2%) were positive for HBV DNA and labeled OBI-positive; their plasma HBV-DNA load was less than 100 IU/ml. Following the phylogenetic analysis, the samples with OBI roughly belonged to genotype D, subtype ayw2 and only two had mutations within the S 'gene's major hydrophilic region (MHR), including T123I, C124F, and P127T. This study reports the prevalence of OBI in the HBsAg-negative HD patients being at a rate of 4.2%, which can be a clinically vital consideration in this region. HBV serologic screening approaches need to be renewed to cover nucleic acid testing in the setting of hemodialysis and all the other high-risk groups associated with it (i.e., blood and organ donors).

Figure 1

(a) Agarose gel electrophoresis of the nested PCR for OBI. C+: positive control; L: 100 bp DNA ladder; C−: negative control; S: OBI-positive samples (227 bp). (b) Agarose gel electrophoresis of the second round of nested PCR for HBsAg amplicon with a length of 700 bp. C+: positive control; L: 100 bp DNA ladder; C−: negative control; S: amplified fragment for sequencing (700 bp).

Figure 2

Representation of five HBsAg sequences (681 nucleotides) acquired from OBI-positive samples. The amino acid substitutions are reported in different colors by the BioEdit program. The top sequence belongs to GQ183486 HBsAg from an Indian genotype D isolate.

Figure 3

The amino acid sequence alignment of the HBV S gene as compared to the reference sequence. The OBI number is shown next to each patient sample separately. A pink box indicates the sequence of “a” determinant. Dots represent the identical amino acids as in the reference sequence, whereas letters represent the variations.