Comparative study of PCR-based Cryptosporidium discriminating techniques with a review of the literature

We identified the species or genotypes of the six Cryptosporidium isolates from patients and C. parvum strain HNJ-1 using the seven previously described species-differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. In addition, we also discussed about the usefulness of these PCR-based protocols on the basis of the reports previously published. Cryptosporidium diagnostic fragment was amplified by PCR with each primer pair, targeting the 18S ribosomal RNA (18SrRNA), Cryptosporidium oocyst wall protein (COWP). Heat shock protein 70 (HSP70), Polythreonine (Poly-T), Thrombospondin related adhesive protein of Cryptosporidium-1 (TRAP-C1), and unknown gene locus, in all isolates from patients and the strain HNJ-1. The RFLP profiles of 18SrRNA, COWP, HSP70, Poly-T, and TRAP-C1 PCR products in all isolates from patients were found to be the same among isolates, and were correspondent to those of C. parvum human genotype. While the RFLP profiles of HNJ-1 were strictly different from those of isolates from patients, and were correspondent to C. parvum cattle genotype. In addition, nucleotide sequences in 18 SrRNA gene of all isolates from patients and HNJ-1 were found to be identical to that of C. parvum, human or cattle genotype, respectively. Therefore, the isolates from patients and HNJ-1 were identified as C. parvum human and cattle genotype, respectively. According to the reports related to the PCR-based protocols applied in the present study, RFLP profiles targeting the HSP70, Poly-T, TRAP-C1 genes had been revealed in only a few species or genotypes, but those of 18SrRNA and COWP genes were in all species and genotypes. However, we supposed that it was difficult to distinguish between human or cattle genotype and other species or genotypes by RFLP profiles of 18SrRNA or COWP because the RFLP profiles of human or cattle genotype were identical or similar to those of other species or genotypes. On the other hand, it has been known that the nucleotide sequences in 18SrRNA or COWP gene are different among Cryptosporidium species and/or genotypes. Therefore, the direct sequencing method targeting the variable regions which can be used to distinguish among Cryptosporidium species, as well as the genotypes within C. parvum in either 18SrRNA or COWP gene is the most useful tool for accurate identification of Cryptosporidium isolates.     

Overview of Regular Dialysis Treatment in Japan as of 31 December 2006

A statistical survey of dialysis patients for the year 2006 was carried out for 4051 medical facilities across Japan, and responses were received from 3985 (98.37%) facilities. There were 264 473 dialysis patients (including 9003 peritoneal dialysis patients) in Japan at the end of 2006, which showed an increase of 6708 (2.6%) from the end of 2005. The number of patients per million population was 2069.9. The crude mortality rate during 2006 was 9.2%. The mean age of the patients who began dialysis (in 2006) was 66.4 years, and the mean age of the entire dialysis population was 64.4 years. The primary renal diseases of the patients who began dialysis were diabetic nephropathy (42.9%), chronic glomerulonephritis (25.6%), and nephrosclerosis (9.4%). Of the 3488 facilities that participated in the survey on the dialysate water quality, 2873 facilities (82.4%) measured the endotoxin concentration in the dialysate; and 1197 facilities (37.1%) out of 3228 measured the bacterial count in the dialysate. The mean hemoglobin concentration in the dialysis population at the end of 2006 was 10.23 ± 1.33 g/dL, which was equal to that at the end of 2005 (10.23 ± 1.37 g/dL). The mean concentration of serum creatinine in 15 853 patients who started dialysis during 2006 was 8.37 ± 3.58 mg/dL. The estimated glomerular filtration rate, which was calculated with formula modified for the Japanese population from the Modification of Diet in Renal Disease (MDRD) Study equation, was 5.46 ± 6.60 mL/min/1.73 m².     

MEFV mutation analysis of familial Mediterranean fever in Japan

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever with serosal inflammation. FMF gene (MEFV) mutations have been identified primarily in patients from Mediterranean populations. Although several clinical cases have been reported in Japan, there have been few reports to date on mutation analysis. We studied FMF patients and their relatives to examine the clinical and genetic features of this disease in the Japanese population. METHODS: Twelve Japanese FMF patients who met the Tel Hashomer criteria and a total of 17 relatives from 5 of 10 families underwent molecular genetic studies to detect MEFV mutations. The characteristics of these Japanese FMF patients and geno-phenotypical correlations were examined. RESULTS: Almost all of our patients had been suffering for a long time from fever of unknown origin and one patient also had systemic amyloidosis. In our 12 FMF patients, we detected the substitutions E84K, L110P, E148Q, R761H and M694I. We also newly diagnosed 2 relatives as having FMF based on clinical symptoms and the existence of FMF mutations. One patient was homozygous for E148Q, the patient with systemic amyloidosis was a homozygote for M694I and 4 patients from 3 families were compound heterozygotes for E148Q and M694I. Three patients in one family were compound heterozygotes for E148Q, L110P and M694I. There were 3 patients who were heterozygous for E84K, L110P-E148Q or M694I and had no other nucleotide changes in the exons of MEFV. On the other hand, 2 relatives who had never experienced symptoms of FMF were homozygous for L110P-E148Q as well as compound heterozygous for E148Q/E148Q-R761H. E148Q and M694I were the most frequently detected substitutions in our study. CONCLUSIONS: MEFV mutations occur in Japanese FMF patients though FMF is rare in Japan. The identification of MEFV mutations could be a reliable diagnostic test for FMF. The results of genetic analyses on 14 Japanese FMF patients in this study revealed that E148Q and M694I are frequent alleles.     

Impact of cytomegalovirus serostatus on outcome of unrelated cord blood transplantation for adults: a single-institute experience in Japan

Cytomegalovirus (CMV) disease is one of the major infectious complications after allogeneic hematopoietic stem cell transplantation (SCT). Several studies have shown that CMV-seropositive patients have a substantial survival disadvantage after bone marrow transplantation (BMT) or peripheral blood SCT (PBSCT). Between August 1998 and February 2006, 101 adult patients underwent myeloablative cord blood transplantation (CBT) from unrelated donors at our institution. Sixteen and 85 patients were CMV-seronegative and CMV-seropositive, respectively, prior to CBT. Outcomes of CBT were compared between CMV-seronegative and CMV-seropositive patients. The cumulative incidences of neutrophil engraftment at 60 d after CBT did not differ between CMV-seronegative and CMV-seropositive patients (100% and 94%, P = 0.09); however, the cumulative incidence of platelet engraftment at 100 d was higher in CMV-seronegative patients than CMV-seropositive patients (100% vs. 86%, P < 0.005). The cumulative incidence of CMV antigenemia at 100 d was lower in CMV-seronegative patients than CMV-seropositive patients (0% vs. 77%, P < 0.001); however, the cumulative incidences of CMV disease did not differ between CMV-seronegative and CMV-seropositive patients (0% vs. 1%, P = 0.84). The probabilities of disease-free survival at 2 yr also did not differ between CMV-seronegative and CMV-seropositive patients (92% vs. 72%, P = 0.16). The outcomes of CBT for CMV-seropositive patients as well as CMV-seronegative patients in our series were favorable. This might be due to effective antiviral therapy for CMV infection. Large-scale studies are needed to determine the impact of recipient CMV serostatus on the outcome of CBT for adults.     

CD19-targeting liposomes containing imatinib efficiently kill Philadelphia chromosome-positive acute lymphoblastic leukemia cells

Patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) have poor prognosis despite intensive therapeutic intervention. Recently, imatinib, a BCR-ABL tyrosine kinase inhibitor, has been proven to be an effective treatment for Ph(+) ALL, but nearly all patients rapidly acquire resistance. High-dose imatinib administration might overcome this resistance; however, systemic toxicities would likely limit this approach. Therefore, a new delivery system allowing for the specific targeting of imatinib is urgently needed. Because almost all Ph(+) ALL cells express CD19 on their surface, we have developed an immunoliposome carrying anti-CD19 antibody (CD19-liposomes). The internalization efficiency of the CD19-liposomes approached 100% in all Ph(+) ALL cells but was very low in CD19(-) cells. The cytocidal effect of imatinib-encapsulated CD19-liposomes (imatinib-CD19-liposomes) on Ph(+) ALL cell lines and primary leukemia cells from patients with Ph(+) ALL was much greater than that of imatinib with or without control liposomes. Importantly, the imatinib-CD19-liposomes did not affect the colony formation of CD34(+) hematopoietic cells, even at inhibitory concentration of free imatinib. Taken together, these data clearly demonstrate that the imatinib-CD19-liposomes induced specific and efficient death of Ph(+) ALL cells. This new therapeutic approach might be a useful treatment for Ph(+) ALL with fewer side effects than free imatinib.     

Human Herpesvirus 6 Variant B Infection in Adult Patients after Unrelated Cord Blood Transplantation

Human herpesvirus 6 variant B (HHV-6B) infection was studied in 23 adult patients who underwent cord blood transplantation (CBT). HHV-6B DNA was detected by quantitative polymerase chain reaction analysis after CBT in the sera from 15 patients (65%) at day 14 or 15 (week 2), from 16 patients (70%) at day 21 or 22 (week 3), and from 3 patients (13%) at day 28 or 29 (week 4). HHV-6B DNAemia was found in none of the 20 patients examined at day 7 or 8 (week 1). The overall incidence of HHV-6B DNAemia reached 87% (20 of 23 patients). This incidence was much higher than after unrelated bone marrow transplantation (19%, P < .0001). In CBT patients, positive HHV-6B DNAemia at week 3 was significantly associated with early skin rash (88% versus 14%, P < .005) and grade II-IV acute graft-versus-host disease (aGVHD) (69% versus 14%, P < .05). In contrast, positive HHV-6B DNAemia at week 2 was associated with neither skin rash nor aGVHD. Prospective large-scale studies are needed to determine the role of HHV-6 infection in CBT patients.     

Unrelated cord blood transplantation after myeloablative conditioning in adults with ALL

We analyzed the disease-specific outcomes of adult ALL treated with cord blood transplantation (CBT) after myeloablative conditioning. Between October 2000 and November 2007, 27 adult patients with ALL were treated with unrelated CBT. All patients received four fractionated 12 Gy TBI and chemotherapy as myeloablative conditioning. The median age was 36 years, the median weight was 57 kg and the median number of nucleated cells was 2.47 x 10(7)/kg. All patients received a single and HLA-mismatched cord blood unit. The cumulative incidence of neutrophil recovery at day 30 and platelet recovery at day 200 was 92.6 and 92.3%, respectively. With a median follow-up of 47 months, the probability of EFS at 5 years was 57.2%. The 5-year cumulative incidence of TRM and relapse was 3.7 and 27.4%, respectively. These results suggest that unrelated CBT after myeloablative conditioning could be safely and effectively used for adult patients with ALL.     

Myeloablative unrelated cord blood transplantation for acute leukemia patients between 50 and 55?years of age: single institutional retrospective comparison with patients younger than 50?years of age

Increasing recipient age is a well-known risk factor for graft-versus-host disease (GVHD) and treatment-related mortality (TRM) and has a negative impact on allogeneic hematopoietic stem cell transplantation. Since the incidence of severe GVHD after cord blood transplantation (CBT) is lower than that after transplants using bone marrow or mobilized peripheral blood grafts from adult cells, we should expect better outcomes from CBT in older patients. To evaluate the feasibility and efficacy of myeloablative unrelated CBT in patients aged between 50 and 55 years, we performed a retrospective comparison of 100 patients with acute leukemia who received cord blood grafts at our institution. Nineteen older patients (median age, 52; range, 50–55) and 81 younger patients (median, 36; range, 16–49) received a myeloablative conditioning regimen including 12 Gy of total body irradiation and chemotherapy. GVHD prophylaxis included cyclosporine with (n = 96) or without (n = 4) methotrexate. There were no significant differences in the incidences of grades II to IV acute GVHD, extensive-type chronic GVHD, TRM, and the probability of overall and disease-free survival between these groups. These results suggest that, in patients with acute leukemia, myeloablative CBT might be as safe and effective in patients aged between 50 and 55 years as in younger patients.