Department of Internal Medicine, 1 Gastroenterology Unit and 2 Nephrology Unit, University of Genoa, Genoa, Italy
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Abstract |
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Methods. Serum samples of 78 patients on maintenance haemodialysis were tested for TTV-DNA, hepatitis G virus (HGV)-RNA, anti-E2, anti-hepatitis C virus (HCV) and HCV-RNA. TTV-DNA was detected by semi-nested PCR using the primers from open reading frame 1 (ORF). HGV-RNA was detected by PCR using specific primers for the NS3 and the 5'-UTR genome regions while anti-E2 were checked by an enzyme immunological test. Anti-HCV was tested by the second generation Chiron RIBA HCV test system. HCV-RNA was evaluated by nested PCR with primers directed to the highly conserved 5' non-coding region of the HCV genome.
Results. TTV prevalence in our patients was 19% (15/78) while the prevalence of HCV and HGV infection proved to be 20 and 15.4%, respectively. Among TTV positive patients HGV co-infection was present in five cases (33%), HCV in six cases (39.9%), while HBV co-infection was not present in any of the patients. Only three patients proved positive for all three viruses. ALT levels were normal in most cases (13/15; 86%). In particular, patients with TTV infection alone showed normal ALT levels and HCV coinfection was found in the two patients with moderate ALT increases.
Conclusions. TTV prevalence in haemodialysed patients is significant though the real clinical impact is still unclear. However, we must keep in mind that the epidemiological relevance of TTV infection is probably underestimated due to the impossibility in detecting the corresponding antibody.
Keywords: TTV-DNA; TTV; HGV-RNA; HGV antibody; hepatitis C virus; haemodialysis
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Introduction |
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The few data available concerning TTV infection among haemodialysis patients show a high prevalence 32% [12].
The aim of our study was to detect TTV-DNA in a group of haemodialysis patients and to evaluate its clinical impact, taking into account co-infection with other hepatitis viruses.
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Patients and methods |
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Methods |
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Viral-RNA extraction
Sera were stored at -80°C within 2 h of withdrawal. Nucleic acids were extracted from 140 µl of serum according to Kit QIAamp HCV (QIAGEN) assay and subsequently resuspended in RNAase-free water. Total RNA was primed with random hexamers and reverse transcription to cDNA was performed at 42°C for 30 min (AMV, Boehringer Mannheim).
HCV-RNA detection
cDNA was amplified by using a nested PCR method with four primers complementary to the highly conserved 5' non-coding region of the HCV genome [13].
HGV-RNA detection
cDNA was subjected to PCR using specific primers (0.2 µM final concentration) for the NS3 and the 5'-UTR genome regions in all serum samples.
5'-UTR region.
The first round of PCR was performed using G1, 5'-AAGCCCCAGAAACCGACGCC-3', as sense primer and G2, 5'-TGAAGGGCGACGTGGACCGT-3', as antisense primer. Nested PCR was performed using G3, 5'-CGGCCAAAAGGTGGTGGATG-3', as sense primer and G4, 5'-GTAACGGGCTCGGTTTAACG-3', as antisense primer [14]. Cycling conditions were: 40 cycles for the first round (consisting of denaturation for 60 s at 94°C, annealing for 60 s at 55°C and extension for 90 s at 72°C) followed by an extension at 72°C for 5 min; 35 cycles for the second round, under the same conditions (DNA Thermal Cycler 480, Perkin-Elmer Cetus).
NS3/helicase region.
The first PCR was performed using G8, 5'-TATGGGCATGGHATHCCYCT-3' (H=A, C or T; Y=C or T) as sense primer and G9, 5'-TCYTTGATGATDGAACTGTC-3' (Y=T or C; D=A, G or T) as antisense primer. Nested PCR was performed using a semi-nested protocol with G8 as sense primer and G11, 5'-TCYTTACCCCTRTAATAGGC-3' (Y=C or T; R=A or G) as antisense primer [14]. Forty cycles were run for the first round (consisting of denaturation for 60 s at 94°C, annealing for 60 s at 57°C and extension for 90 s at 72°C) followed by an extension at 72°C for 5 min; 35 cycles were performed for the semi-nested PCR, under the same conditions (DNA Thermal Cycler 480, Perkin-Elmer Cetus).
PCR products were subjected to electrophoresis on 2% agarose gel (Agarose LE, Genenco). The expected size for the PCR products to be derived from NS3 and 5'-UTR were 140 and 204 bp, respectively with reference to the standard molecular weight (174 RF DNA/HaeIII).
Sequence analysis (Amplicycle Sequencing Kit, Perkin-Elmer) was performed using G4 and G11 as sequencing primers for both genomic areas in the two PCR positive haemodialysis patients to confirm the specificity of the PCR amplified products. The nucleotide sequences of the 5'-UTR region showed 100% homology with the HGV sequence (GeneBank accession number U44402). The sequence of the NS3 region showed 95% homology.
Detection of anti-E2
The serum qualitative determination of IgG antibodies to the HGV E2-antigen was performed by the enzyme immunological test (µPLATE Anti Hgenv, Boehringer Mannheim).
Detection of TTV
DNA was extracted from 200 µl serum using QIAmp Blood kit (QIAGEN Ltd, Crawley, UK) and resuspended in 50 µl elution buffer. TTV-DNA was amplified by semi-nested PCR with TTV-specific primers derived from two conserved regions according to the published sequences [8].
The first round of PCR was performed using primer TTV1 (sense primer 5'-ACAGACAGAGGAGAAGGCAAYATG -3') and primer TTV3 (antisense primer 5'-CTGGCATTTTACCATTTCCAAAGTT-3'). Amplification was for 35 cycles at 94°C for 45 s, 58°C for 30 s and 72°C for 45 s, followed by 4 min at 72°C. The second round was performed with another sense primer TTV2 (5'-GGCAACATGTTATGGATAGACTGG-3') and the same antisense primer TTV3 using 1 µl of the first round PCR. Amplification was obtained by the same method. The amplicons were electrophoresed in 1% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. Sequence by Sanger method using inner primer was used to confirm the specificity of the amplification products.
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Results |
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Patients with TTV infection alone showed normal ALT levels while HCV co-infection was present in the two patients with moderate ALT elevation.
We found a high prevalence of history of transfusion among TTV-DNA +ve patients (8/15; 53.3%) when compared with TTV-DNA -ve patients (16/63; 25.3%; P<0.05; 2 test). No statistically significant differences were present between these groups for what concern age, sex, time on haemodialysis, liver function test or prevalence of HCV or HGV infection (Table 1
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Discussion |
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A possible explanation for this difference may be the different spreading of infection between the two geographic areas. Moreover, taking into account the existence of different previously described TTV genotypes the geographic, genotypic prevalence may underestimate the real diffusion of infection in our area [1518].
The percentage of patients who have undergone multiple transfusions was higher in the TTV positive group (eight out of 15 patients, 53.3%) than in the TTV negative group (16 out of 63 patients, 25.3%) suggesting that the parenteral route may play an important role in transmitting the virus. In fact when haemodialysed patients were grouped according to their history of multiple transfusions, TTV infection was present in eight of the 24 polytransfused patients (33.3%) and in seven of the 54 non-transfused ones (12.9%). Moreover, the TTV infection alone was present in four polytransfused patients (16.6%), but only in three (5.5%) of the non-transfused patients. We did not find any difference related to age, time on haemodialysis, liver function test or prevalence of HCV or HGV infection between TTV +ve or -ve patients, as previously reported [1219,20].
TTV infection in the non-transfused patients might be related to dialysis or to a non-parenteral way as has previously been described. In fact, the discovery of TTV excretion in the faeces suggests that the infection may be transmitted by faecaloral route [21]. Regarding possible sexual transmission, a low percentage of TTV infection in groups of subjects at high risk for this kind of transmission was observed [22]. Moreover, the importance of non parenteral transmission of TTV is supported by the higher percentage of infection than other hepatitis viruses among blood donors [23,24].
As far as the clinical impact of TTV infection is concerned, our data show that the patients infected with TTV alone had normal ALT levels while the biochemical alterations in the TTV positive cases with moderate ALT increase may be accounted for by HCV co-infection, as already observed [25,26]. Presently the lack of tests to detect antibodies against TTV, may cause an underestimation of the real diffusion of TTV infection and, thus, its epidemiological impact. No data are available regarding the prevalence of TTV infection in a control population in our area. However, in another group of patients at risk of parenteral transmission (anti-HCV positive, but without history of transfusion), we observed a low prevalence of TTV infection (four out 65 patients, 6.2% unpublished data). Furthermore, recent data from an Italian survey on blood donors reports a prevalence of TTV infection of 5±1.9% in our country [27].
In conclusion, although TTV prevalence in haemodialysed patients is significant, the real clinical impact is still unclear. In fact our data did not show that when TTV alone is present it induces liver function tests alteration. However, it must be taken into account that the epidemiological relevance of TTV infection is probably underestimated due to the difficulty in detecting the corresponding antibody.
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Notes |
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References |
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