Outbreak of infections by hepatitis B virus genotype A and transmission of genetic drug resistance in patients coinfected with HIV-1 in Japan.

The major routes of hepatitis B virus (HBV) infection in Japan has been mother-to-child transmission (MTCT) and blood transfusion. However, HBV cases transmitted through sexual contact are increasing, especially among HIV-1-seropositive patients. To understand the molecular epidemiology of HBV in HBV/HIV-1 coinfection, we analyzed HBV genotypes and HIV-1 subtypes in HBV/HIV-1-coinfected patients at Nagoya Medical Center from 2003 to 2007. Among 394 HIV-1-infected Japanese men having sex with men (MSM) who were newly diagnosed during the study period, 31 (7.9%) tested positive for the hepatitis B virus surface antigen. HBV sequence analyses were successful in 26 cases, with 21 (80.7%) and 5 (19.3%) cases determined as genotypes A and C, respectively. Our finding that HBV genotype A was dominant in HIV-1-seropositive patients alerts clinicians to an alternative outbreak of HBV genotype A in the HIV-1-infected MSM population and a shift in HBV genotype from C to A in Japan. The narrow genetic diversity in genotype A cases suggests that genotype A has been recently introduced into the MSM population and that sexual contacts among MSM were more active than speculated from HIV-1 tree analyses. In addition, we found a lamivudine resistance mutation in one naïve case, suggesting a risk of drug-resistant HBV transmission. As genotype A infection has a higher risk than infection with other genotypes for individuals to become HBV carriers, prevention programs are urgently needed for the target population.

Mutation of the beta1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly.

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We identified the first TUBB1 mutation, R318W, in a patient with congenital macrothrombocytopenia. The patient was heterozygous for Q43P, but this single-nucleotide polymorphism (SNP) did not relate to macrothrombocytopenia. Although no abnormal platelet beta1-tubulin localization/marginal band organization was observed, the level of beta1-tubulin was decreased by approximately 50% compared with healthy controls. Large and irregular bleb protrusions observed in megakaryocytes derived from the patient's peripheral blood CD34(+) cells suggested impaired megakaryocyte fragmentation and release of large platelets. In vitro transfection experiments in Chinese hamster ovary (CHO) cells demonstrated no incorporation of mutant beta1-tubulin into microtubules, but the formation of punctuated insoluble aggregates. These results suggested that mutant protein is prone to aggregation but is unstable within megakaryocytes/platelets. Alternatively, mutant beta1-tubulin may not be transported from the megakaryocytes into platelets. W318 beta1-tubulin may interfere with normal platelet production, resulting in macrothrombocytopenia.

High performance liquid chromatography using UV detection for the simultaneous quantification of the new non-nucleoside reverse transcriptase inhibitor etravirine (TMC-125), and 4 protease inhibitors in human plasma.

Etravirine (TMC-125, ETV) is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) that demonstrates potent activity against NNRTI-resistant strains of human immunodeficiency virus type-1 (HIV-1). Thus, ETV has been used in combination with ritonavir-boosted protease inhibitor (PI) and integrase inhibitor for therapy-experienced HIV-1-infected patients. On the other hand, as ETV is a substrate and inducer of cytochrome P450 3A4 (CYP3A4), ETV may induce metabolism of PI and alter the concentrations of co-administered PIs. In order to ensure optimal drug efficacy and prevention of resistance, it is essential to monitor plasma concentrations of ETV and PIs. Here we describe the application of HPLC with UV detection for the simulataneous assay of ETV and 4 PIs, darunavir (DRV), atazanavir (ATV), ritonavir (RTV) and lopinavir (LPV). In this study, the calibration curve of each drug was linear with the average accuracy ranging from 93.6 to 110.9%. Both intra- and interday coefficients of variation for each drug were less than 11.6%. The mean recovery of all drugs ranged from 88.0 to 97.5%. The limit of quantification was 0.04, 0.04, 0.04, 0.05 and 0.07 microg/ml for ETV, DRV, ATV, RTV and LPV, respectively. These results demonstrate that our HPLC-UV method can be used for routine determination of plasma concentrations of ETV and 4 PIs in clinical settings.

Safety and efficacy of rasburicase (SR29142) in a Japanese phase II study.

The purpose of this study was to investigate the safety profile of SR29142 when administered as a single agent both prior to chemotherapy and during treatment, and to compare the efficacy of SR29142 administered at two dose levels in adult Japanese patients with leukemia or lymphoma. During this open-label, multicenter, phase II study, patients received SR29142 for 5 days, administered at either 0.15 or 0.20 mg/kg per day. Chemotherapy was started 4­24 h after the first infusion of SR29142. The primary end-point was overall response rate, defined as the normalization of plasma uric acid to 7.5 mg/dL or less, from 48 h after the first infusion to 24 h after the last infusion of SR29142. SR29142-related adverse events including hypersensitivity (allergic) reactions were assessed. Overall, 50 patients received SR29142 at either 0.15 mg/kg per day (n = 25) or 0.20 mg/kg per day (n = 25) followed by chemotherapy. The overall response rate was 100.0% (95% confidence interval, 86.3­100.0%) with 0.15 mg/kg and 96.0% (95% confidence interval, 79.6­99.9%) with 0.20 mg/kg. Both dose levels of SR29142 were equally effective at reducing plasma uric acid levels. In six patients, seven drug-related adverse events of grade 1/2 occurred before chemotherapy. SR29142-related, hypersensitivity-associated reactions occurred in three patients, and rash, anorexia, application site pain and pyrexia occurred in one patient each; only five patients (10%) showed anti-SR29142 antibodies by day 29. In conclusion, SR29142 is effective at reducing plasma uric acid levels with a tolerable safety profile as a single agent both prior to chemotherapy and during treatment.

Early central nervous complications after umbilical cord blood transplantation for adults.

Early central nervous complications (CNS) are significant after allogeneic stem cell transplantation; however, the clinical characteristics of early CNS complications have not yet been well described. The medical record of 77 patients who underwent cord blood transplantation (CBT) between March 2001 and November 2005, at 8 centers of the Nagoya Blood and Marrow Transplantation Group were retrospectively reviewed. The preparative regimen included myeloablative CBT (n = 31) or reduced-intensity (RI)-CBT (n = 46). Of the 77 patients, 10 (13%) developed early CNS complications. Causes included Cyclosporine encephalopathy (n = 5), tacrolimus encephalopathy (n = 2), thrombocytic microangiopathy (n = 1), and unknown (n = 3). The median time of onset was 19 days (range: 2-58 days). All of the 10 patients developed impaired consciousness. Seizures developed in 6 patients. Early CNS complications spontaneously subsided in 3 patients. Three patients responded to cyclosporine or tacrolimus discontinuation. The remaining 4 patients died within 30 days of developing of early CNS complications. No relationship was detected between the preparative regimen and the onset of early CNS complications, while an HLA disparity showed borderline significance (hazard ratio, 3.24; 95% confidential interval, 0.94-11.20; P = .06). Early CNS complications are a significant problem after CBT, and the clinician has to be aware of the possibility of these complications.

Trend of drug-resistant HIV type 1 emergence among therapy-naive patients in Nagoya, Japan: an 8-year surveillance from 1999 to 2006.

We studied the emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) with major amino acid mutations in 402 therapy-naive patients at Nagoya Medical Center, Japan, between 1999 and 2006. The mean prevalence of drug-resistant HIV-1 was 6.7% (range, 2.3-10.0%; n = 27). HIV-1 variants with protease inhibitor (PI)-resistant mutations alone were most frequently found (3.5%, n = 14), followed by those with nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutations alone (1.7%, n = 7). Variants with nucleoside reverse transcriptase inhibitor (NRTI)-resistant mutations alone were sporadically found (1.0%, n = 4). A variant possessing both NRTI- and PI-resistant mutations was detected in one patient (0.2%) and a variant possessing both NNRTI- and PI-resistant mutations was identified in another patient (0.2%). In addition, another 17 variants (4.2%, n = 17) with only 215-revertant mutations (T215C/D/G/L/S) that can easily reconvert to the nucleoside analogue-associated mutation of T215Y/F were found. The 402 viruses were phylogenetically analyzed, revealing three independent clusters comprising PI-resistant variants with the M46I or L90M mutation, NNRTI-resistant variants with the K103N mutation, and 215-revertant variants. The PI-resistant and 215-revertant strains have been spreading since 2000, and the NNRTI-resistant strain has started spreading since 2003. The nature of the epidemic and information for successfully blocking the spread of drug-resistant HIV-1 were clarified in this study.

Hematopoietic stem cell transplantation for acute promyelocytic leukemia in second or third complete remission: a retrospective analysis in the Nagoya Blood and Marrow Transplantation Group.

Acute promyelocytic leukemia (APL) is the most curable subtype of acute myeloid leukemia. Second complete remission (CR2) can be easily achieved with several therapeutic options even after relapse. However, the optimal strategy to treat APL in CR2 is still controversial. We retrospectively compared the outcome of autologous (auto) and allogeneic (allo) hematopoietic stem cell transplantation (HSCT) for patients with APL in CR2 or CR3. Fifteen patients received auto and 13 received allo HSCT between 1999 and 2004 at eight hospitals belonging to the Nagoya Blood and Marrow Transplantation Group. Four-year disease-free survival (DFS) and overall survival (OS) for autografted patients were 68.9 and 75.8%, whereas those for allografted patients were 46.2 (P = 0.350) and 46.2% (P = 0.185), respectively. Three autografted patients and one allografted patient relapsed, and one autografted patient and five allografted patients died without leukemia relapse. Among 14 autografted patients who were evaluated for MRD with molecular analysis, relapse occurred in one with positive MRD (n = 2) and two with negative MRD (n = 12). These data suggest that auto HSCT is very effective for APL in CR2 or CR3, and may be preferable to allo HSCT for a portion of patients. Prospective studies are required to define the role of auto HSCT in the treatment of relapsed APL.

Identification and characterization of the first large deletion of the MYH9 gene associated with MYH9 disorders.

MYH9 disorders are autosomal dominant macrothrombocytopenias with leukocyte inclusion bodies. Single point mutations in the protein-coding sequence of the MYH9 gene are the most common cause. So far no large gene deletion/insertion and splicing defects have been reported. Conventional DNA sequencing of each MYH9-coding exon showed no abnormalities in a patient. Reverse transcription- polymerase chain reaction (PCR) amplification and sequencing of neutrophil mRNA identified an inframe deletion of exon 25. Further long-range PCR amplification of genomic DNA revealed a deletion of 1220 nucleotides including entire exon 25. Immunoblot analysis showed a small, abnormal protein in neutrophils but not in platelets. This is the first report of a large deletion of the MYH9 gene leading to the development of MYH9 disorders.

[Clinical features and course of 5 cases with HIV encephalopathy].

Clinical features and courses of 5 cases with HIV encephalopathy were reported. The subjects were among the patients registered as HIV patients at the Nagoya Medical Center, between 1996 and 2005. There were 458 patients with HIV infection including 127 cases of AIDS. All patients suffered from severe immunological deficiency when HIV encephalopathy developed. Other opportunistic infections had also occurred in three patients. HIV encephalopathy was one of the presenting manifestations of HIV infection in four patients, and no patients had received antiretroviral therapy. HAART improved motor disturbance and their ADL became independent except for one case. Improvements in neuropsychological examination scores were noted in all cases. Recovery from psychiatric symptoms, however, was incomplete. Four patients could not work, and 3 needed psychological treatment due to behavioral abnormalities. HIV encephalopathy is not a lethal disease but the functional prognosis was very poor. New therapy is needed for HIV encephalopathy.

Successful efavirenz dose reduction in HIV type 1-infected individuals with cytochrome P450 2B6 *6 and *26.

BACKGROUND: Efavirenz (EFV) is metabolized primarily by cytochrome P450 2B6 (CYP2B6), and high plasma concentrations of the drug are associated with a G-->T polymorphism at position 516 (516G-->T) of CYP2B6 and frequent central nervous system (CNS)-related side effects. Here, we tested the feasibility of genotype-based dose reduction of EFV. METHODS: CYP2B6 genotypes were determined in 456 human immunodeficiency virus type 1 (HIV-1)-infected patients who were receiving EFV treatment or were scheduled to receive EFV-containing treatment. EFV dose was reduced in CYP2B6 516G-->T carriers who had high plasma EFV concentrations while receiving the standard dosage (600 mg). EFV-naive homozygous CYP2B6 516G-->T carriers were treated with low-dose EFV. In both groups, the dose was further reduced when plasma EFV concentration remained high. RESULTS: CYP2B6 516G-->T was identified in the *6 allele (found in 17.9% of our subjects) and a novel allele, *26 (found in 1.3% of our patients). All EFV-treated CYP2B6 *6/*6 and *6/*26 carriers had extremely high plasma EFV concentrations (>6000 ng/mL) while receiving the standard dosage. EFV dose was reduced to 400 mg for 11 patients and to 200 mg for 7 patients with persistently suppressed HIV-1 loads. EFV-containing treatment was initiated at 400 mg in 4 CYP2B6 *6/*6 carriers and one *6/*26 carrier. Two of them still had a high plasma EFV concentration while receiving that dose, and the dose was further reduced to 200 mg, with successful HIV-1 suppression. CNS-related symptoms improved with dose reduction in 10 of the 14 patients, although some had not been aware of the symptoms at initial dosage. CONCLUSIONS: Genotype-based EFV dose reduction is feasible in CYP2B6 *6/*6 and *6/*26 carriers, which can reduce EFV-associated CNS symptoms.

No observable correlation between central nervous system side effects and EFV plasma concentrations in Japanese HIV type 1-infected patients treated with EFV containing HAART.

The present study assessed the relationship between central nervous system (CNS) side effects and plasma concentrations of efavirenz (EFV) in Japanese HIV-1-infected patients. Subjects consisted of 69 HIV-1-infected patients (57 therapy-naive and 12 therapy-experienced patients) being treated using EFV in combination with other antiretroviral agents at the outpatient HIV clinic. Successful virological treatment was achieved in 61 patients. Eight patients discontinued EFV containing therapy because CNS symptoms did not resolve (four patients), EFV-specific mutations were detected (two patients), or skin rash was observed (two patients). Mean EFV plasma concentration for 61 effectively treated patients, measured at 15 h postdosing, was 2.42 microg/ml (range: 0.78-6.82 microg/ml). This EFV concentration range contributed to suppressed viral load in these Japanese patients. Adverse CNS effects were observed in 19 patients soon after therapy onset. These effects disappeared within 1 month except for four patients who suffered severe CNS side effects. Mean EFV plasma concentrations were not significantly different between subjects with (2.45 +/- 1.08 microg/ml) and without (2.42 +/- 1.40 microg/ml) CNS side effects. We concluded no correlation existed between the plasma EFV concentration and the emergence of CNS side effects in Japanese HIV-1-infected patients. Further investigations, enforced with the drug concentration measurement at earlier time points and more appropriate assessment of CNS symptoms, are required.

Drug-resistant HIV-1 prevalence in patients newly diagnosed with HIV/AIDS in Japan.

The increasing prevalence of drug-resistant HIV transmission has become a critical epidemic in the world today. Studies in developed countries reported 8-27% of newly diagnosed HIV/AIDS patients are infected by drug-resistant strains. To determine the prevalence of drug-resistant HIV-1 among newly diagnosed cases in Japan, eight HIV/AIDS clinical centers, three public health laboratories and the National Institute of Infectious Diseases conducted a nationwide survey. Between January 2003 and December 2004, 575 newly diagnosed HIV/AIDS patients with both acute and chronic infections were enrolled in the study. Twenty-three cases, including three recently infected patients, were infected with HIV-1 having major drug-resistance mutations, including M41L, D67N, L100I, K103N, V106A, M184I, M184V, L210W, and revertant mutations at the 215 codon in reverse transcriptase and M46I in protease encoding regions. In this newly diagnosed population, we also clarified the prevalence of hepatitis virus coinfection, which was 8.8% for HBV and 4.3% for HCV. In conclusion, the drug-resistant transmission rate was 4.0% in Japan. Although this rate is significantly lower than that of other developed countries, this rate almost reaches the threshold at which baseline genotypic resistance testing would be cost-effective for all infected persons before initiating therapy.

[Clinical course of the disease and the level of WT1 mRNA in 191 patients with acute myeloid leukemia (AML): joint research by 23 institutions in Japan].

We evaluated the clinical course of acute myeloid leukemia (AML) and the levels of WT1 mRNA in 191 AML patients. Of 114 previously untreated patients with AML, 107 cases were positive for WT1 mRNA (93.9% : 107/114). WT1 mRNA expression-levels declined to below 50 copies/microg RNA ("negative") after remission was achieved in all 66 patients who achieved remission and 84.8% (47/54) cases were "negative" at the end of the follow-up periods. On the other hand, WT1 mRNA was expressed in 87.0% of non-remission cases (47/54), maintaining 50 copies/microg of RNA or higher ("positive"). In all 29 cases who relapsed during the follow-up observation period after achieving remission, WT1 mRNA levels declined transiently approximately around the time of achieving remission and then rose again when the disease relapsed. Moreover, we determined the time of elevation of WT1 mRNA in 29 relapsed cases. In 79.3% of relapsed cases (23/29), WT1 mRNA levels rose above 200 copies/microg RNA, 43 days (median) before the diagnosis of "relapse". Given the percent of the correct diagnosis, WT1 mRNA at 200 copies/microg RNA appeared to be a reasonable cut-off level for early detection of AML-relapse. The WT1 mRNA level reflected the clinical condition. Taken together, these findings indicate that WT1 mRNA levels allow us to detect the presence of so-called "minimal residual disease" (leukemic cells) that cannot be detected by morphological examination. Besides these promising data, this kit is suitable for routine monitoring of AML because this kit utilizes peripheral blood as a test specimen, reducing the patient's burden at the time of collection of clinical samples as compared with bone marrow aspirate.

Retrospective study on the impact of hepatitis B and hepatitis C virus infection on hematopoietic stem cell transplantation in Japan.

We performed a retrospective survey in 62 hematopoietic cell transplantation (HCT) centers in Japan in which all HCTs performed between 1986 and 1998 were reviewed, and those involving hepatitis B virus surface antigen (HBsAg)-positive donors were identified. One hundred and thirty-five patients who underwent allogeneic HCT (alloHCT) were studied for complications related to hepatitis B virus (HBV) or hepatitis C virus (HCV). The median follow-up period was 24 months. Positivity for HBsAg was observed in 32 patients (24%) throughout the study. Twenty-six of the 32 patients were HBsAg carriers before alloHCT, whereas the remaining 6 became HBsAg(+) after alloHCT. Forty-two recipients were anti-HBs antibody (HBsAb)-positive, and 58 recipients (43%) were HCV Ab(+). Eleven of 26 (42%) HBsAg(+) recipients survived between >4 and >119 months. Six of 26 cases received transplants from HBsAg(+) donors, and, although they had not developed acute graft-versus-host disease, 4 of 6 died of hepatic and renal failure within 10 months after HCT. After transplantation, 5 patients showed serologic evidence of HBV reactivation, whereas 4 patients showed evidence of an immune response to HBV. Viral reactivation occurred during the tapering of the immunosuppressive agent. However, 3 of 5 were alive at the time of this report, suggesting that reactivation is not directly correlated with severe liver dysfunction. Seventeen patients (13%) of 135 recipients developed hepatic failure. Eight (47%) of 17 were diagnosed with fulminant hepatitis and 5 (29%) with veno-occlusive disease (VOD). VOD was observed in 12% of both HBsAg(+) and HCVAb(+) patients. In this study, the relatively high incidence of HBV events occurred after alloHCT, and, therefore, we should consider a protocol for active immunization of donors and recipients against HBV. Moreover, although the presence of HBV or HCV is not a contraindication for alloHCT, we recommend a careful follow-up of recipients after transplantation, especially during immunosuppression tapering.

A novel splice site mutation in intron C of PROS1 leads to markedly reduced mutant mRNA level, absence of thrombin-sensitive region, and impaired secretion and cofactor activity of mutant protein S.

Protein S (PS) is a member of the vitamin K-dependent protein family containing similar gamma-carboxyglutamic acid (Gla) domains, although only PS has a thrombin-sensitive region (TSR), which is located between the Gla domain and the first epidermal growth factor-like domain. In this study, a novel PROS1 mutation was identified at the last nucleotide in intron C (c.260-1G>A) in a patient suffering from recurrent deep vein thrombosis associated with PS deficiency. To investigate the molecular mechanisms of PS deficiency caused by the novel PROS1 mutation, we characterized the mutant mRNA, and the secretion and function of the mutant PS molecule associated with the mutation. RT-PCR was used to detect the aberrant mRNA in the patient's platelets, the amount of which was markedly reduced and lacked the region corresponding to exon 4 coding the TSR of the PS molecule. The recombinant mutant PS lacking the TSR (TSR-lack PS) showed a markedly reduced transient expression/secretion level, 37.9% of that of wild-type (WT) PS. Activated protein C (APC) cofactor activity assay showed that TSR-lack PS had no cofactor activity. Moreover, binding assays of monoclonal antibodies recognizing the PS Gla domain and the Gla residues indicated that the bindings of TSR-lack PS to both of these antibodies were clearly weaker than those of WT PS. These findings suggest that the novel mutation leading to the absence of the TSR not only affected the secretion of mutant PS, but was also responsible for impairment of the Gla domain conformation required for the gamma-carboxylation to express APC cofactor activity.

HIV-2 CRF01_AB: first circulating recombinant form of HIV-2.

BACKGROUND: Five HIV-2-seropositive cases were recently identified in Japan, outside the HIV-2 endemic area of West Africa. To clarify the molecular epidemiology of HIV-2 in Japan, we analyzed sequences of these cases in detail. METHODS: HIV-2 genetic groups were determined by gag and env sequences. For suspected recombinant isolates, the genetic structure was determined by full-length genomic analyses. To understand the history and evolution of HIV-2 recombinant isolates, we estimated the time of most recent common ancestor by Bayesian Markov chain Monte Carlo method. RESULTS: Three isolates were determined as recombinants of groups A and B, and their mosaic genome structures were identical with that of 7312A, a recombinant isolate reported in 1990 from Côte d'Ivoire. Our 3 isolates and 7312A fulfilled the criteria for determining a circulating recombinant form (CRF). These isolates were verified by the Los Alamos HIV sequence database as the first CRF of HIV-2, HIV-2 CRF01_AB. The mean time of most recent common ancestor of CRF01_AB was estimated as between 1964 and 1973, several decades after the estimated emergence of HIV-2. CONCLUSIONS: We recently identified HIV-2 CRF01_AB cases in Japan. This ectopic observation of the virus outside its original endemic area suggests an ongoing global spread of HIV-2 CRF01_AB.

The clinical characteristics of CD7+ CD56+ acute myeloid leukemias other than M0.

Immunological phenotyping of acute leukemia have provided enormous and important information for the classification and lineage determination of leukemia. Forty-nine patients with CD7(+) CD56(+) acute myeloid leukemia (AML) were analyzed. There were 17 patients of M0, which corresponded to myeloid/NK cell precursor acute leukemia, and 32 patients of AML other than M0 (9 each for M1 and M2, one for M3, 3 for M4, 4 for M5 and 6 for M7). Age distribution was similar between these two groups, but CD7(+) CD56(+) M0 showed significant male predominance than CD7(+) CD56(+) M1-M7 (M:F = 15:2 vs. 15:17, P = 0.006). The disease localization and the hematological manifestations were different, showing fewer white blood cell counts and circulating leukemic blasts, less anemia, less thrombocytopenia and more frequent extramedullary involvement in M0 group. The prognosis was poor in both groups, and there was no statistical difference. These findings suggest that extramedullary involvement of myeloid/NK cell precursor acute leukemia is not directly derived from the presence of CD7 and CD56 antigens on leukemic cells. The poor prognosis of CD7(+) CD56(+) M1-M7 suggests that this phenotype may act as a prognostic factor for AML, but this should be confirmed in further studies.