In 1997, a novel DNA virus, designated TT virus (TTV), was reported to be associated with increased aminotransferase levels in the sera of patients with posttransfusion hepatitis of unknown origin (1). Although there is clear evidence for transmission of TTV through blood and blood products, the natural route of its transmission is still unknown. The high TTV DNA prevalence among healthy subjects and the presence of the virus in stool and in saliva suggests that TTV might be transmitted nonparenterally (2,3). Some investigators have observed a significantly higher prevalence of TTV in patients with cryptogenetic chronic liver disease and fulminant hepatitis compared with blood donors (4,5). However, the results obtained by use of the most sensitive primers from 5´NCR (5´ noncoding region) that show a high incidence of TTV in the general population and blood donors suggested that the prevalence of TTV infection reported previously may have been significantly underestimated (6,7). The aims of this study were 1) to evaluate the frequency of TTV infection in children with a high risk of virus transmission by the parenteral route (blood transfusion) compared with healthy children and blood donors, and 2) to correlate the association of TTV with the biochemical data of liver disease.
Thirty-seven thalassemic outpatients (21 boys) of Pediatric Clinic of “Policlinico Umberto I” (Rome) and 36 healthy children (19 boys) without a history of remarkable diseases and who were seen for checkups in the same clinic in the same period were included in the study. Median age was 12 years (range, 7–20 years) for thalassemic patients and 10 years (range, 1–16 years) for healthy children (P = not significant). One hundred blood donors, with a median age of 38 (range, 19–45 years), 83 of whom were men and were seen in the same period in the blood transfusion center of “Policlinico Umberto I” (Rome), were also included in the study.
All thalassemic patients received regular blood transfusions every 7 to 35 days, according to the clinical conditions of their hematologic disease, and iron chelation therapy according to current protocols. All pediatric and young adult patients and healthy children had a history of vaccination against hepatitis B virus (HBV). The hepatitis B surface antigen (HbsAg) tested negative in all blood donors. All healthy children and blood donors were negative for anti–hepatitis C virus (HCV). Transaminase levels were normal in blood donors and in healthy children.
Anti-HCV was determined by third-generation enzyme-linked immunoassay-enhanced SAVe, (Ortho, New Jersey). The presence of HCV RNA was determined by polymerase chain reaction (PCR) in 5´NCR, as previously described (8).
Sample Preparation and PCR-Based Amplification of TTV DNA
The TTV detection in sera was assessed using a PCR-based assay. The TTV DNA was extracted from 200 μL serum using commercial silica columns (QiaAmp Kit, Qiagen, Hilden, Germany), following manufacturer instructions. The nucleic acid was eluted in a final volume of 50 μL water. Ten microliters of extracted DNA were used for TTV amplification by semi-nested PCR using a first set of primers that recognize an internal sequence into N22 (9) and by nested PCR with a second set of primers amplifying a sequence within 5´NCR (6).
Cloning and Sequence Analysis
The N22 amplicons obtained after second-round PCR were cloned into vector pCR II TOPO (Invitrogen BV, NV Leek, The Netherlands) and then sequenced on both strands, using primersT7 and SP6 as sequencing primers and the DNA Sequencing Kit (Perkin Elmer, California) by the ABI 373A automated sequencer (PE Applied Biosystem, Shelton, CT). The nucleotide sequence was determined on at least three clones for each PCR product.N22 sequences (210 nucleotides, positions 1939 to 2148, according to the sequence of the prototype TTV isolate, accession number AB008394) from random clones were compared with each other and with the sequence of prototype TTV (9,10).
The prevalence of TTV DNA in our populations, detected with the two different sets of primers, is reported in Table 1. The first set of primers showed TTV DNA in 73% of thalassemic patients, in 8% of healthy children, and in 5% of healthy blood donors. With the second set of primers, the prevalence of TTV DNA was, respectively, 100% in thalassemic patients, 44.5% in healthy pediatric patients, and 87% in healthy blood donors. All individuals who tested positive for TTV by the first set of primers were also positive by the second primer set. The respective age-specific prevalence of TTV detected by the second set of primers in healthy children was 58.8% (10 of 17) for children 1 to 8 years and 31.5% (6 of 19) for children older than 8 years. The presence of TTV in healthy children and in blood donors was not associated with altered transaminase levels (see Patients and Methods). Correlations between transaminase levels and HCV markers in TTV-positive thalassemic patients are reported in Table 2. Anti-HCV antibodies were found in 24 of 37 thalassemic patients (64%), and 14 of them were also HCV RNA positive. Eighteen of 37 thalassemic patients showed altered transaminase levels at the time of TTV detection. Of these, 15 were positive for anti-HCV and 10 were also HCV RNA positive (mean alanine transaminase value, 61 ± 45 U/L). At the time of TTV detection, slightly altered transaminase levels (less than 1.5 times the upper reference limit) were shown in eight HCV RNA–negative patients. Six months later, normal transaminase levels were found, even though TTV DNA was still detected.
Fifteen amplicons from randomly selected thalassemic patients (10 samples) and from unselected blood donors (5 samples) were subjected to TTV DNA sequence analysis. The results confirmed the specificity of the amplified products (data not shown). Alignment of each PCR product with the N22 sequence of the prototype TTV DNA and with genotypes and subtypes reported in the literature (10,11) accomplished using a Pileup package (Wisconsin Package, Genetics Computer Group, Cambridge, UK), suggested the classification of our isolates into genotypes G1 and G2 (data not shown). Analysis of four clones detected in the same serum of three thalassemic patients (patients 2, 7, and 9) showed the contemporaneous presence of two distinct TTV variants. The predominant clones are indicated with “a,” the others with “b” (Fig. 1). The homology between sequences “a” and “b” was high in two patients (92.9%), but minimal for one patient (65.3%). No different viral variants were observed in all the TTV-positive samples from blood donors.
Several studies have shown different prevalence values of TTV in different populations (4,5,12). Because of the marked heterogeneity of the nucleotide sequences of TTV isolates, particularly those included in the G2 group, it is not certain that the primers used in those studies can detect all the possible variants of the virus. In effect, the set of primers suggested by Takahashi et al. (T801 and T935) (6) was found to be 10 to 100 times more sensitive than the set of primers (NG061 and NG063) described by Okamoto et al. (10), used previously in most of the prevalence studies. In fact, the high prevalence values shown with the most sensitive set of primers in our blood donors are in agreement with the prevalence values found by other studies in blood donors and in the healthy general population (80–92%) (6,7). The discrepant values of prevalence found in healthy children and in blood donors using the two sets of primers (8% vs. 44.5% and 5% vs. 87%, respectively) confirm that the choice of PCR primers is critical in the detection of TTV and, consequently, in the evaluation of transmission routes and in establishing the clinical significance of this viral agent. Although the prevalence of TTV infection detected using the second set of primers in healthy children is lower compared with thalassemic patients and with blood donors (44.5% vs. 100% and 87%), it is higher compared with the prevalence reported in a recent study performed on healthy children (21%) from a different geographic area with the same set of primers (13). The different age prevalence values found in our healthy children confirm other reports showing that TTV infection is common in healthy children and more frequently in early childhood. Recent studies have reported a high frequency of postnatal transmission of TTV that might be attributed to close contact with a TTV-infected mother (14–17). Other epidemiologic studies may help to understand the high prevalence values and define other transmission routes in blood donors and the healthy general population.
In agreement with other data (13,16,18), in our population of healthy children and blood donors, in which a high prevalence of TTV was found using the second sets of primers, TTV seems to exist frequently without evident alteration of the biochemical parameters. In the TTV DNA–positive thalassemic patients without other known hepatitis virus infection, the observed slight alteration of serum transaminase levels might correlate with iron-induced hepatic damage or with the presence of TTV variants or TT-related viruses (not detected by our sets of primers) or other unidentified blood-borne pathogens.
Only one TTV strain was detected in the sera of blood donors, as previously reported (9). In this study, different TTV variants were observed in some thalassemic patients. In one serum, two different isolates with very low homology (65.3%) were detected, clearly indicating the coexistence of two different genotypes. The contemporaneous presence of distinct strains of TTV in the same patient belonging to high-risk groups from various countries has already been reported and suggested to have occurred by superinfection with a different TTV by successive transfusions (5,11). In the two patients for whom 92.9% homology was detected between the two isolates, the possibility of an evolutionary event during the virus replication cannot be ruled out.
In conclusion, in the light of the results obtained with the new most sensitive primers (5´NCR) the prevalence of TTV in polytransfused children is similar to that detected in blood donors. Our results show that TTV can be detected in healthy children of all ages. The presence of TTV seems to have no clinical significance.
The authors thank Mrs. Gina Mauro for her editorial help.
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