Influence of Primers on the Detection of TT Virus DNA by Polymerase Chain Reaction

We read with great interest the article by Irving et al. [1] who showed different prevalence rates of TT virus (TTV) in patients with chronic hepatitis C by 3 PCR methods with primers deduced from distinct regions of the TTV genome [2–4]. TTV is a nonenveloped and single-stranded DNA virus isolated from a patient with posttransfsuion hepatitis of unknown cause [2, 5]. Because TTV has a circular genomic structure [6–8], it would be classified into the Circoviridae family. DNA of TTV is detected in blood donors and hepatitis patients over the world [2, 3, 9], whereas the hepatitis-inducing capacity of TTV needs to be established. TTV has a wide range of sequence divergence by which it is classified into at least 16 genotypes [10]; they are separated by an evolutionary distance (number of nucleotide substitutions per site) >0.30. The nucleotide sequence in the untranslated region (UTR) is more conserved than that in the coding region [8, 10]. Hence, primers deduced from different areas of the TTV genome crucially influence the detection of TTV DNA by polymerase chain reaction (PCR) [4, 10]. Should certain genotypes of TTV be associated with liver disease, DNA of such genotypes would need to be detected by PCR with primers specific for them. We determined TTV DNA by 2 distinct PCR methods in Japanese blood donors with and without elevated alanine aminotransferase (ALT) levels. PCR with heminested primers (NG059/NG063 and NG061/NG063), deduced from the N22 region (N22 PCR), is specific for the detection of TTV DNA of genotypes from 1 to 6 [2, 10]. The other PCR with nested primers (NG133/NG147 and NG134/NG132), deduced from well-conserved areas in the UTR, detected TTV DNA of all known genotypes from 1 to 16 [10]. Blood donors were selected who had not received transfusion and who were negative for serum markers of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus type 1. Of the 1128 blood donors, 547 had elevated ALT levels (61–301 U/L; mean ± SD, 87.4 ±33.6 U/L) and the remaining 581 had normal ALT levels (6–45 U/L; mean ± SD, 19.9 ± 7.7 U/L). Table 1 compares the detection of TTV DNA by N22 PCR and UTR PCR in blood donors with and without elevated ALT levels, stratified by age. TTV DNA was detected by N22 PCR in 139 (25.4%) of 547 blood donors with elevated ALT levels at a frequency significantly higher (P < .0001; χ2 test) than that in 91 (15.7%) of 581 donors with normal ALT levels. The prevalence of TTV DNA increased with age in blood donors either with or without elevated ALT levels. By contrast, TTV DNA was detected by UTR PCR at a high frequency (⩾93%) in all age groups with no difference in prevalence between the blood donors with and without elevated ALT levels. TT virus DNA in blood donors detected by 2 distinct polymerase chain reaction (PCR) methods. These results, taken along with those of Irving et al. [1], indicate that the selection of PCR primers crucially influences the detection of TTV DNA. Furthermore, these results suggest that TTV of restricted genotypes, detected by N22 PCR [2, 5], would be associated with liver damage among blood donors.