Norihiko Akamatsu

Possible origin of adult T-cell leukemia/lymphoma cells from human T lymphotropic virus type-1-infected regulatory T cells.

Adult T‐cell leukemia/lymphoma (ATLL) is a lymphoproliferative disorder caused by human T lymphotropic virus type 1 (HTLV‐I). Although ATLL cells display an activated helper/inducer T‐cell phenotype, CD4+ and CD25+, they are known to exhibit strong immunosuppressive activity. As regulatory T cells (Treg cells) express CD4+ and CD25+ molecules and possess potent immune response suppressive activity, we investigated a possible link between ATLL cells and Treg cells. In primary ATLL cells, the expression levels of the Treg cell marker molecules Foxp3 and glucocorticoid‐induced tumor necrosis factor receptor family related protein (GITR) were significantly higher than in those from healthy adults. Furthermore, ATLL cells are unresponsive in vitro to concanavalin A stimulation and suppress the proliferation of normal T cells. GITR mRNA expression was induced by the HTLV‐I transactivator Tax, and GITR promoter analyses revealed that this induction depends on the κB site from −431 bp to −444 bp upstream of the putative transcription site. Taken together, ATLL cells may originate from HTLV‐I‐infected Treg cells, and GITR seems to be involved in the progression to ATLL. (Cancer Sci 2005; 96: 527–533)

Rapid, Simple, and Accurate Detection of K-ras Mutations From Body Fluids Using Real-Time PCR and DNA Melting Curve Analysis

The K-ras oncogene is one of the most useful genetic markers in screening for the presence of cancers because it is largely involved in tumorigenesis. However, most mutationdetection techniques are generally unsuitable for routine use, especially due to their timeconsuming, labor-intensive, and sensitive natures. Accordingly, we attempted to establish a new technique for analysis of K-ras alterations at codons 12 and 13 from body fluid specimens with tumor DNA using realtime polymerase chain reaction (PCR) with melting curve analysis (MCA). In this PCRMCA method, K-ras was easily genotyped by melting temperatures (Tm) that differ by 3.6°C to 11.6°C with an acceptable reproducibility of Tm. The shape of the melting curve gave easier interpretation. The detection sensitivity was at least 10 -3 . The entire process of the test was completed within 4 hours, saving 7 to 8 hours when compared to the current PCR-restriction fragment length polymorphism (RFLP) analysis. The examination of the MCA method using 38 clinical samples revealed the better detection rate (32% versus 24%) and diagnostic efficiency (100% versus 92%) compared with that of the PCR-RFLP. In conclusion, this small scale but high throughput method is acceptable for clinical use to analyze K-ras alterations from body fluids and plasma DNA, including small tumor DNA.

Rapid and high-resolution detection of IgH gene rearrangements using PCR and melting curve analysis

The analytical methods of Southern blot hybridization (SBH) and the polymerase chain reaction (PCR) for complementarity determining region‐3 (CDR3) are fundamental for detecting IgH gene rearrangement. However, there are problems stemming from the characteristics of both methods; especially, the long turn around time (TAT) because of the complex process in the SBH, and the low analytical sensitivity for amplicons in the PCR. Thus, to improve the PCR procedure, we investigated the application of detecting the clonal amplicons based on the different melting Temperature (Tm) in internal melting domains corresponding to the CDR3 hypervariable region. Our new protocol is based on the combination of a LightCycler Technology with high‐speed amplification, and Idaho‐Technology with rapid and high‐resolution melting curve analysis (MCA), designated PCR‐MCA. This method can provide the results within 3 h with an analytical sensitivity of 10−3. The diagnostic sensitivity and specificity relative to the results documented with the SBH analysis were 89.2% and 100%, respectively. This indicates that the new protocol of PCR‐MCA is acceptable for clinical testing; especially, PCR‐MCA is relevant in terms of the rapid and sensitive detection of IgH clonality within amplicons.

Antimicrobial susceptibility and molecular characteristics of methicillin-resistant Staphylococcus aureus in a Japanese secondary care facility

Methicillin-resistant Staphylococcus aureus (MRSA) is prevalent in Japan, and the Staphylococcus cassette chromosome mec (SCCmec) type II is common among hospital-acquired MRSA isolates. Information pertaining to MRSA characteristics is limited, including SCCmec types, in primary or secondary care facilities. A total of 128 MRSA isolates (90 skin and soft tissue isolates and 38 blood isolates) were collected at a secondary care facility, Kawatana Medical Center, from 2005 to 2011. Antimicrobial susceptibility testing for anti-MRSA antibiotics and molecular testing for SCCmec and virulence genes (tst, sec, etb, lukS/F-PV) were performed. Strains positive for lukS/F-PV were analyzed by multilocus sequence typing and phage open-reading frame typing. SCCmec typing in skin and soft tissue isolates revealed that 65.6% had type IV, 22.2% had type II, 8.9% had type I, and 3.3% had type III. In blood isolates, 50.0% had type IV, 47.4% had type II, and 2.6% had type III. Minimum inhibitory concentrations, MIC(50)/MIC(90), against vancomycin, teicoplanin, linezolid, and arbekacin increased slightly in SCCmec II isolates from skin and soft tissue. MICs against daptomycin were similar between sites of isolation. SCCmec type II isolates possess tst and sec genes at a greater frequently than SCCmec type IV isolates. Four lukS/F-PV-positive isolates were divided into two clonal patterns and USA300 was not included. In conclusion, SCCmec type IV was dominant in blood, skin, and soft tissue isolates in a secondary care facility in Japan. Because antimicrobial susceptibility varies with the SCCmec type, SCCmec typing of clinical isolates should be monitored in primary or secondary care facilities.

Fluoroquinolone resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a Japanese tertiary hospital: silent shifting to CTX-M-15-producing K. pneumoniae

Purpose. Fluoroquinolone resistance (FQ‐r) in extended‐spectrum β‐lactamase (ESBL) producers is an urgent health concern in countries where ESBL‐producing K. pneumoniae (ESBL‐Kpn) is prevalent. We investigated FQ‐r in Japan where ESBL‐Kpn is less prevalent. Methodology. Clinical ESBL‐Kpn isolates from 2011 to 2013 were collected in Nagasaki University Hospital. The ESBL genotypes included CTX‐M‐15, and the mechanisms of FQ‐r through plasmid‐mediated quinolone resistance (PMQR) and mutations in quinolone resistance‐determining regions (QRDRs) were examined. Clonality was analysed by enterobacterial repetitive intergenic consensus (ERIC)‐PCR and multi‐locus sequence typing was performed on selected isolates. Results/Key findings. Thirty ESBL‐Kpn isolates, including seven levofloxacin‐resistant isolates, were obtained from different patients. An increase in CTX‐M‐15‐producing strains was observed during the study period (0/11 in 2011, 3/8 in 2012, and 5/11 in 2013). PMQR was detected in 53.3% of the isolates and aac‐(6′)‐Ib‐cr was the most common (36.7 %). ST15 was observed in 60.0% of the isolates, and for the most predominant ERIC‐PCR profiles, 62.5% of the isolates possessed the CTX‐M‐15 genotype and 71.4% were levofloxacin‐resistant. Levofloxacin‐resistance was significantly more common in CTX‐M‐15 isolates (62.5 %) compared to non‐CTX‐M‐15 isolates (9.1 %). Three QRDR mutations and aac(6′)‐Ib‐cr, but not qnrB and qnrS, were significantly enriched in the CTX‐M‐15 isolates (100.0 %) compared to the non‐CTX‐M‐15 isolates (13.6 %). Conclusion. Cumulatively, these results indicate that the epidemic strain, the CTX‐M‐15‐producing K. pneumoniae ST15, is covertly spreading even when ESBL producers are not prevalent. Monitoring these epidemic strains and ESBLs in general is important for quickly identifying health crises and minimizing future risks from FQ‐r ESBL‐Kpn.

Sequential Washing with Electrolyzed Alkaline and Acidic Water Effectively Removes Pathogens from Metal Surfaces

Removal of pathogenic organisms from reprocessed surgical instruments is essential to prevent iatrogenic infections. Some bacteria can make persistent biofilms on medical devices. Contamination of non-disposable equipment with prions also represents a serious risk to surgical patients. Efficient disinfection of prions from endoscopes and other instruments such as high-resolution cameras remains problematic because these instruments do not tolerate aggressive chemical or heat treatments. Herein, we develop a new washing system that uses both the alkaline and acidic water produced by electrolysis. Electrolyzed acidic water, containing HCl and HOCl as active substances, has been reported to be an effective disinfectant. A 0.15% NaCl solution was electrolyzed and used immediately to wash bio-contaminated stainless steel model systems with alkaline water (pH 11.9) with sonication, and then with acidic water (pH 2.7) without sonication. Two bacterial species (Staphylococcus aureus and Pseudomonas aeruginosa) and a fungus (Candida albicans) were effectively removed or inactivated by the washing process. In addition, this process effectively removed or inactivated prions from the stainless steel surfaces. This washing system will be potentially useful for the disinfection of clinical devices such as neuroendoscopes because electrolyzed water is gentle to both patients and equipment and is environmentally sound.

Active Surveillance of Methicillin-Resistant Staphylococcus aureus Using a Fully Automated Molecular Test in an Emergency Medical Center

The prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) are important, particularly in emergency units. The active surveillance of MRSA was prospectively performed at the emergency medical center of Nagasaki University Hospital. After obtaining nasal swab specimens, a fully automated molecular test (FAMT) and a culture-screening method were utilized for MRSA detection. A total of 150 patients were enrolled in the study, and 366 nasal swab specimens were obtained. MRSA was detected by culture in 11 (7.3%) patients including one new acquisition and by the FAMT in 34 (22.7%) patients including 13 new acquisitions. The sensitivity, specificity, positive predictive value, and negative predictive value of the FAMT at the patient level were 86.7, 85.2, 39.4, and 98.3%, respectively, when compared with the culture-based results. An FAMT can effectively detect MRSA colonization, which may remain undetected with the conventional method, and it may be useful in detecting newly acquired MRSAs.

Performance evaluation of BD Phoenix™, an automated microbiology system, for the screening of IMP-producing Enterobacteriaceae

BD Phoenix™ is an automated bacterial identification and susceptibility testing system. Here, its performance in screening IMP-producing Enterobacteriaceae was evaluated. The system identified 97.8% of IMP producers as being nonsusceptible to imipenem or meropenem, which was higher than that identified by the broth microdilution method (91.3%, imipenem; 41.3%, meropenem).

The Rapid Induction of Carbapenem-Resistance in an Aeromonas dhakensis Blood Isolate

Meropenem-susceptible and -resistant Aeromonas dhakensis isolates from blood cultures of a fatal case of septicemia were analyzed. The two isolates were homologous and gene expression of metallo-β-lactamase in the resistant strain was upregulated. Physicians should be aware of the possibility of the induction of carbapenem-resistance, following the use of carbapenems in the treatment of Aeromonas infection.

Usefulness of a comprehensive PCR-based assay for human herpes viral DNA in blood mononuclear cell samples.

Human herpes virus (HHV) is well known to reactivate in immunocompromised situations and plays an immunomodulatory role leading to indirect effects through viral replication to the host. The aim of this study was to determine the laboratory and clinical relevance of a comprehensive PCR-based assay for detecting eight HHVs, which are lymphotropic and cause disease in humans. Using 176 samples collected from 146 specimens of peripheral blood, 12 skin nodules, 11 lymph nodes and 7 others of patients who were suspected to have adult T cell leukemia (ATL), the PCR-based assay was validated to simultaneously detect one or more herpes viral DNA with two consensus primer sets. Although most samples were seropositive for either of the HHVs, only 50% of them were positive for either herpes viral DNA with EBV in 76%, HHV-6 in 14% and VZV in 9%. Furthermore, such a herpes viral DNA positive status was not always associated with clinical symptoms relating to the virus, implying active replication in the blood cells, but being asymptomatic. HHV-8 viral DNA, although Kaposis sarcoma has been reported to be complicated with ATL, was not demonstrable. HHV-6B was detected only in HTLV-1 healthy carriers and ATL patients, and may imply a co-factor role with HTLV-1. This PCR-based assay provides a herpes viral infectious status compensatory for virus-serology and is a clinically relevant laboratory test, serving as a screening marker for active infection.