Influence of TT virus co-infection on IFN-beta therapy in patients with chronic hepatitis C.

Although results of IFN alpha therapy in chronic hepatitis C (C-CH) patients co-infected with TT virus (TTV) have been reported, no results of IFN beta therapy or IFN beta and alpha combination therapy have been reported. In this study, we retrospectively investigated whether co-infection with TTV affects the results of IFN therapy by using stored sera from 60 C-CH patients co-infected with TTV who underwent IFN beta therapy or IFN beta and alpha combination therapy. The stored sera were from 29 complete responders, 10 incomplete responders, and 21 non-responders, and they were used for qualitative and quantitative analysis of HCV RNA, HCV genotype analysis, and qualitative and quantitative analyses of TTV DNA. TTV DNA was detected in 23 (38.3%) of the 60 C-CH sera. The TTV DNA-positive rate was 17.2% among the complete responders to IFN therapy, versus 58.1% in the incomplete responders and non-responders, and the difference was significant (p < 0.01). While the complete response prediction rate based on two factors, HCV RNA level and HCV genotype, was 80.8% (21/26) in the C-CH patients, the prediction rate based on three factors, these two factors plus TTV DNA, was higher, 90.0% (18/20). It was concluded that determination of HCV RNA concentration, HCV genotype, and TTV DNA, before IFN beta therapy or IFN beta and alpha combination therapy is useful for predicting the results of treatment of C-CH patients.

Selection of more appropriate PCR primer pairs for improved efficiency in detecting Norwalk-like virus (NLV) RNA.

A pair of primers, NV35 and NV36, and another pair of primers, NV81 and NV82/SM82, are commonly used for polymerase chain reaction (PCR) detection of Norwalk-like virus (NLV) genome RNA sequences in authorized test laboratories in Japan. However, the efficiency of NLV genome RNA detection with these primer pairs has been less than satisfactory. In the present study, we attempted to establish more appropriately matched primer pairs for improved detection of NLV genome RNA sequences using a combination of primers including NV35, NV36, NV81, NV82/SM82, SR33, and SRs (a mixture of 4 primers SR46, SR48, SR50, and SR52). We also evaluated appropriate primers for improved reverse transcription of NLV genome RNA. Stool samples used for detection of NLV included 18 samples collected from NLV-infected patients who ingested oysters (group 1) and 13 samples collected from those who did not ingest oysters (group 2). Reverse transcription of RNA genome with primer NV35 was less efficient compared with that with primer SR33 or NV81. When PCR products obtained with NV35 and NV36 as a pair of primers were subjected to gel electrophoresis, a strong extra band was detected compared with those obtained with other primer pairs. Since this extra band may represent heterodimeric or homodimeric hybrids, or intramolecular hybrids derived from these primers, this template-independent hybridization could lower the efficiency of primer-dependent polymerase reaction. Of 18 primer pairs, a pair of NV81 and SRs provided the best detection of PCR products following reverse transcription of NLV RNA with SR33 or NV81. The detection rate was 61% for both reverse transcription with SR33 and that with NV81. After reverse transcription using SR33 as a primer, nested PCR using a pair of NV81 and SRs following primary PCR using a pair of NV81 and NV82/SM82 increased the detection rate to 89% in group 1 and 100% in group 2.