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671 REACTIVATION OF HEPATITIS C IN A PATIENT WITH ADVANCED HUMAN IMMUNODEFICIENCY VIRUS INFECTION
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671 REACTIVATION OF HEPATITIS C IN A PATIENT WITH ADVANCED HUMAN IMMUNODEFICIENCY VIRUS INFECTION E.C. Thomson1 , V.M. Fleming3 , J. Main1,2 , M. McClure1 , P. Karayiannis2 . 1 Infectious Diseases, 2 Hepatology, Imperial College, London, 3 Peter Medawar Building for Pathogen Research, Oxford University, Oxford, UK E-mail: [email protected] Background and Aims: A 38 year old man with a diagnosis of human immunodeficiency virus (HIV-1) infection developed acute hepatitis C (HCV) infection (genotype 1a) in January 2003. The patient was treated with pegylated interferon a and ribavirin for one year. Six months after completion of treatment, the HCV RNA remained undetectable by PCR and the ALT was normal. In May 2006 his CD4 count dropped to 106 x106 cells/ml and he required treatment for Pneumocystis jeroveci pneumonia. In October 2006, his previously normal ALT rose to 133 iu/L and HCVRNA was detectable once again. He denied any episodes of unprotected sex or contact with his previous partner, or any other high-risk exposure to HCV. We aimed to investigate whether this was due to reinfection or reactivation of virus. Methods: Archived plasma samples taken from the patient and ten similar HCV and HIV positive patients were analysed for the presence of HCV RNA by nested PCR designed to amplify the hypervariable region-1 (HVR-1) of HCV. The HVR-1 PCR products were purified and ligated into the cloning vector TOPO-4, and transformed in E coli (TOP-10) cells. Twenty colonies were selected from kanamycin-containing agar plates and grown overnight in selection medium. DNA was extracted and sequenced and neighbour-joined phylogenetic trees constructed using MEGA 4.0 software. Results: Branches derived from patient HVR-1 sequences from February 2004 and April 2007 clustered closely together showing that they were closely related. Conclusions: This is the first description of reactivation of hepatitis C infection in an HIV-positive immunosuppressed individual. We hypothesise that the original virus was present in the liver and/or other sanctuary sites, such as peripheral blood mononuclear cells or the central nervous system, and that the cell-mediated immune response had prevented emergence of virus in plasma until the patient became immunosuppressed as a consequence of advanced HIV infection. 672 SCREENING OF OCCULT HBV INFECTION IN BLOOD DONORS IN HONG KONG USING NUCLEIC ACID TESTING D.K.H. Wong1 , C.L. Lai1 , J. Fung1 , C.K. Lee2 , C.K. Lin2 , I.F.N. Hung1 , D. But1 , A. Hsu1 , P. Chan1 , T.K. Cheung1 , F. Fung1 , J.C.H. Yuen1 , J. Young1 , V. Ngai1 , M.F. Yuen1 . 1 Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, 2 Red Cross Transfusion Service, Hong Kong, China E-mail: [email protected] Background and Aims: Post-transfusion hepatitis B infection can still occur with HBsAg-negative blood donors. This is believed to be caused by occult HBV infection, defined by detectable HBV DNA in HBsAgnegative subjects. We aimed to determine the prevalence of occult HBV infection in blood donors in Hong Kong with high endemicity for chronic hepatitis B. Patients and Methods: From January 2006 to September 2007, we prospectively collected serum samples from 10,000 blood donors at the Hong Kong Red Cross Transfusion Service. Sera were screened by the Cobas TaqScreen MPX Test in the s201 system (Roche diagnostics). In subjects with initial positive results, occult HBV status was confirmed by the Artus HBV PCR [lower limit of detection (LLOD): 6.4 copies/mL] (Qiagen), Cobas TaqMan (LLOD: 69.8 copies/mL) (Roche diagnostics),
and an in-house real-time PCR tests (LLOD: 26 copies/mL). Genuine HBV DNA positive result was defined as having 2 out of 3 positive HBV DNA signals by the Artus test. Results: We identified 61 HBsAg-negative, s201-positive cases. Artus HBV test revealed that 43 cases had 2 consecutive negative PCR signal, while 7 cases had only 1 positive PCR result out of 3 tests. Eleven cases had 2 positive HBV DNA results out of 3 tests (range: 0.7−53.9 copies/mL). Thus 11 out of 10,000 donors (0.11%) had confirmed occult HBV infection. Six samples were tested with Cobas TaqMan test and in-house real-time PCR. HBV DNA was undetectable in all 6 samples by the Cobas TaqMan test and detectable in 1 sample by the in-house real-time PCR test. At the time of writing, 5 subjects (3 male, 2 female) underwent liver biopsies. One female subject had normal histology and the other female had mild necroinflammation. Two male subjects had mild to moderate fibrosis, and one male subject had moderate steatosis. Conclusions: The prevalence of occult HBV infection was 0.11% among blood donors in Hong Kong. Due to the extremely low HBV DNA levels in these subjects, it is difficult to accurately diagnose occult HBV infection. Liver histology however showed that some subjects had developed fibrosis and other mild hepatic changes. 673 EVALUATION OF DUAL PRIMING OLIGONUCLEOTIDE (DPO)-BASED MULTIPLEX PCR FOR HBV YMDD MUTANTS IN COMPARISON TO RESTRICTION FRAGMENT MASS POLYMORPHISM (RFMP) AND TRUGENE HBV GENOTYPING H.Y. Woo1 , H.S. Park1 , B.I. Kim2 , J.W. Yun2 , H.J. Kim2 , Y.K. Cho2 , W.K. Jeon2 , Y.J. Kim3 , J.K. Kim4 . 1 Department of Laboratory Medicine, 2 Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, 3 Department of Laboratory Medicine, Masan Samsung Hospital, Sungkyunkwan University School of Medicine, Masan, 4 Seegene Institute of Life Science, Seoul, South Korea E-mail: [email protected] Background and Aims: A novel Dual Priming Oligonucleotide (DPO) primer was developed to detect single nucleotide polymorphisms (SNP) or mutations in a one step PCR assay. We evaluated the usefulness of the DPO-based multiplex PCR to detect lamivudine resistant HBV mutants in comparison with TRUGENETM HBV genotyping kit and the restriction fragment mass polymorphism (RFMP). Methods: The sera from 44 chronic hepatitis B patients were analyzed for the presence of mutations at codons 180 and 204 by performing DPObased multiplex PCR, RFMP, and TRUGENE. Results: Overall concordance rate among three assays was 40.9% (18/44). Concordance rates between multiplex PCR and RFMP or TRUGENE were 61.4% (27/44) and 47.8% (21/44), respectively. DPO-based multiplex-PCR and RFMP revealed additional mutants than other two methods in ten and five patients, respectively, while TRUGENE could not. Especially, multiplex PCR disclosed the cause of high viral load by identifying mutant virus in one patient. Conclusions: DPO-based multiplex PCR was a highly sensitive method to identify minor mutant populations and could be a practical tool in monitoring of lamivudine resistance.
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671 REACTIVATION OF HEPATITIS C IN A PATIENT WITH ADVANCED HUMAN IMMUNODEFICIENCY VIRUS INFECTION E.C. Thomson1 , V.M. Fleming3 , J. Main1,2 , M. McClure1 , P. Karayiannis2 . 1 Infectious Diseases, 2 Hepatology, Imperial College, London, 3 Peter Medawar Building for Pathogen Research, Oxford University, Oxford, UK E-mail: [email protected] Background and Aims: A 38 year old man with a diagnosis of human immunodeficiency virus (HIV-1) infection developed acute hepatitis C (HCV) infection (genotype 1a) in January 2003. The patient was treated with pegylated interferon a and ribavirin for one year. Six months after completion of treatment, the HCV RNA remained undetectable by PCR and the ALT was normal. In May 2006 his CD4 count dropped to 106 x106 cells/ml and he required treatment for Pneumocystis jeroveci pneumonia. In October 2006, his previously normal ALT rose to 133 iu/L and HCVRNA was detectable once again. He denied any episodes of unprotected sex or contact with his previous partner, or any other high-risk exposure to HCV. We aimed to investigate whether this was due to reinfection or reactivation of virus. Methods: Archived plasma samples taken from the patient and ten similar HCV and HIV positive patients were analysed for the presence of HCV RNA by nested PCR designed to amplify the hypervariable region-1 (HVR-1) of HCV. The HVR-1 PCR products were purified and ligated into the cloning vector TOPO-4, and transformed in E coli (TOP-10) cells. Twenty colonies were selected from kanamycin-containing agar plates and grown overnight in selection medium. DNA was extracted and sequenced and neighbour-joined phylogenetic trees constructed using MEGA 4.0 software. Results: Branches derived from patient HVR-1 sequences from February 2004 and April 2007 clustered closely together showing that they were closely related. Conclusions: This is the first description of reactivation of hepatitis C infection in an HIV-positive immunosuppressed individual. We hypothesise that the original virus was present in the liver and/or other sanctuary sites, such as peripheral blood mononuclear cells or the central nervous system, and that the cell-mediated immune response had prevented emergence of virus in plasma until the patient became immunosuppressed as a consequence of advanced HIV infection. 672 SCREENING OF OCCULT HBV INFECTION IN BLOOD DONORS IN HONG KONG USING NUCLEIC ACID TESTING D.K.H. Wong1 , C.L. Lai1 , J. Fung1 , C.K. Lee2 , C.K. Lin2 , I.F.N. Hung1 , D. But1 , A. Hsu1 , P. Chan1 , T.K. Cheung1 , F. Fung1 , J.C.H. Yuen1 , J. Young1 , V. Ngai1 , M.F. Yuen1 . 1 Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, 2 Red Cross Transfusion Service, Hong Kong, China E-mail: [email protected] Background and Aims: Post-transfusion hepatitis B infection can still occur with HBsAg-negative blood donors. This is believed to be caused by occult HBV infection, defined by detectable HBV DNA in HBsAgnegative subjects. We aimed to determine the prevalence of occult HBV infection in blood donors in Hong Kong with high endemicity for chronic hepatitis B. Patients and Methods: From January 2006 to September 2007, we prospectively collected serum samples from 10,000 blood donors at the Hong Kong Red Cross Transfusion Service. Sera were screened by the Cobas TaqScreen MPX Test in the s201 system (Roche diagnostics). In subjects with initial positive results, occult HBV status was confirmed by the Artus HBV PCR [lower limit of detection (LLOD): 6.4 copies/mL] (Qiagen), Cobas TaqMan (LLOD: 69.8 copies/mL) (Roche diagnostics),
and an in-house real-time PCR tests (LLOD: 26 copies/mL). Genuine HBV DNA positive result was defined as having 2 out of 3 positive HBV DNA signals by the Artus test. Results: We identified 61 HBsAg-negative, s201-positive cases. Artus HBV test revealed that 43 cases had 2 consecutive negative PCR signal, while 7 cases had only 1 positive PCR result out of 3 tests. Eleven cases had 2 positive HBV DNA results out of 3 tests (range: 0.7−53.9 copies/mL). Thus 11 out of 10,000 donors (0.11%) had confirmed occult HBV infection. Six samples were tested with Cobas TaqMan test and in-house real-time PCR. HBV DNA was undetectable in all 6 samples by the Cobas TaqMan test and detectable in 1 sample by the in-house real-time PCR test. At the time of writing, 5 subjects (3 male, 2 female) underwent liver biopsies. One female subject had normal histology and the other female had mild necroinflammation. Two male subjects had mild to moderate fibrosis, and one male subject had moderate steatosis. Conclusions: The prevalence of occult HBV infection was 0.11% among blood donors in Hong Kong. Due to the extremely low HBV DNA levels in these subjects, it is difficult to accurately diagnose occult HBV infection. Liver histology however showed that some subjects had developed fibrosis and other mild hepatic changes. 673 EVALUATION OF DUAL PRIMING OLIGONUCLEOTIDE (DPO)-BASED MULTIPLEX PCR FOR HBV YMDD MUTANTS IN COMPARISON TO RESTRICTION FRAGMENT MASS POLYMORPHISM (RFMP) AND TRUGENE HBV GENOTYPING H.Y. Woo1 , H.S. Park1 , B.I. Kim2 , J.W. Yun2 , H.J. Kim2 , Y.K. Cho2 , W.K. Jeon2 , Y.J. Kim3 , J.K. Kim4 . 1 Department of Laboratory Medicine, 2 Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, 3 Department of Laboratory Medicine, Masan Samsung Hospital, Sungkyunkwan University School of Medicine, Masan, 4 Seegene Institute of Life Science, Seoul, South Korea E-mail: [email protected] Background and Aims: A novel Dual Priming Oligonucleotide (DPO) primer was developed to detect single nucleotide polymorphisms (SNP) or mutations in a one step PCR assay. We evaluated the usefulness of the DPO-based multiplex PCR to detect lamivudine resistant HBV mutants in comparison with TRUGENETM HBV genotyping kit and the restriction fragment mass polymorphism (RFMP). Methods: The sera from 44 chronic hepatitis B patients were analyzed for the presence of mutations at codons 180 and 204 by performing DPObased multiplex PCR, RFMP, and TRUGENE. Results: Overall concordance rate among three assays was 40.9% (18/44). Concordance rates between multiplex PCR and RFMP or TRUGENE were 61.4% (27/44) and 47.8% (21/44), respectively. DPO-based multiplex-PCR and RFMP revealed additional mutants than other two methods in ten and five patients, respectively, while TRUGENE could not. Especially, multiplex PCR disclosed the cause of high viral load by identifying mutant virus in one patient. Conclusions: DPO-based multiplex PCR was a highly sensitive method to identify minor mutant populations and could be a practical tool in monitoring of lamivudine resistance.