Real-world treatment patterns and clinical outcomes in patients with AML in Japan who were ineligible for first-line intensive chemotherapy.

Acute myeloid leukemia (AML) predominantly affects elderly adults, and its prognosis worsens with age. Treatment options for patients in Japan ineligible for intensive chemotherapy include cytarabine/aclarubicin ± granulocyte colony-stimulating factor (CA ± G), azacitidine (AZA), low-dose cytarabine (LDAC), targeted therapy, and best supportive care (BSC). The country's aging population and the evolving treatment landscape are contributing to a need to understand treatment pathways and associated outcomes. This retrospective chart review evaluated outcomes in patients across Japan with primary/secondary AML who were ineligible for intensive chemotherapy and began first-line treatment or BSC between 01/01/2015 and 12/31/2018. The primary endpoint was overall survival (OS); secondary endpoints included progression-free survival (PFS) and healthcare resource utilization (HRU). Of 199 patients (58% > 75 years), 121 received systemic therapy (38 CA ± G, 37 AZA, 7 LDAC, 39 other) and 78 received BSC. Median OS was 5.4, 9.2, 2.2, 3.8, and 2.2 months for CA ± G, AZA, LDAC, other systemic therapy, and BSC, respectively; median PFS was 3.4, 7.7, 1.6, 2.3, and 2.1 months, respectively. HRU rates were uniformly high, with > 80% patients hospitalized in each cohort. The poor clinical outcomes and high HRU among Japanese AML patients who are ineligible for intensive chemotherapy highlight an unmet need for novel therapies.

Accumulation of staphylococcal Panton-Valentine leukocidin in the detergent-resistant membrane microdomains on the target cells is essential for its cytotoxicity.

The mechanisms for the cytotoxicity of staphylococcal Panton-Valentine leukocidin (PVL), a pore-forming toxin consisting of LukS-PV and LukF-PV, in human immune cells are still unclear. Because LukS-PV binds to ganglioside GM1, a constituent of detergent-resistant membrane microdomains (DRMs) of the plasma membrane, the role of DRMs in PVL cytotoxicity was examined in human polymorphonuclear neutrophils (PMNs), monocytes, HL-60 cells, and THP-1 cells. PVL binding capacities in HL-60 and THP-1 cells were higher than those in PMNs and monocytes; however, the PVL concentration to obtain more than 80% cell lysis in HL-60 cells was 10 times higher than that in PMNs and PVL even at such concentration induced < 10% cell lysis in THP-1 cells. After incubation of PMNs with LukS-PV, more than 90% of LukS-PV bound to the detergent-soluble membranes. Subsequent incubation with LukF-PV at 4 °C induced the accumulation of more than 70% of PVL components and 170- to 220-kDa complex formation in DRMs in an actin-independent manner. However, only 30% of PVL was found, and complex formation was under detectable level in DRMs in HL-60 cells. PVL did not accumulate in DRMs in THP-1 cells. Our observations strongly indicate that PVL accumulation in DRMs is essential for PVL cytotoxicity.

Reduction of overall Helicobacter pylori colonization levels in the stomach of Mongolian gerbil by Lactobacillus johnsonii La1 (LC1) and its in vitro activities against H. pylori motility and adherence.

The effects of Lactobacillus johnsonii La1 (LC1) on Helicobacter pylori colonization in the stomach were investigated. H. pylori colonization and gastritis in LC1-inoculated Mongolian gerbils were significantly less intense than those in the control animals. LC1 culture supernatant (>10-kDa fraction) inhibited H. pylori motility and induced bacterial aggregation in human gastric epithelial cells, suggesting the potential of clinical use of LC1 product.

Virulence genotypes and drug resistance of Helicobacter pylori from Vladivostok, Russia: another feature in the Far East.

Helicobacter pylori in Vladivostok, Far Eastern Russia, was investigated during 2004 to 2009. The genotype cagA(+) vacA(+) (s1/m1 or m2) accounted for 74.7%, with cagA(-) vacA(+) (s2/m2) at 11.2%. The CagA EPIYA type was mainly Western ABC, with minor types (ABCCC and novel AAABC) or non-Western/non-East Asia type (AB). Regarding drug resistance, metronidazole resistance was the highest, with a marked decrease in 6 years (from 71.4% to 30.8%); in contrast, levofloxacin and clarithromycin resistance increased. The data indicate that in Vladivostok, H. pylori was mainly the Western (not East Asian) type and dynamic changes in drug resistance occurred during 6 years.

Helicobacter pylori eradication by sitafloxacin-lansoprazole combination and sitafloxacin pharmacokinetics in Mongolian gerbils and its in vitro activity and resistance development.

A total of 293 strains of Helicobacter pylori, including strains resistant to levofloxacin, clarithromycin, metronidazole, or amoxicillin, were examined for in vitro susceptibility to 10 antimicrobial agents. Among these agents, sitafloxacin (a fluoroquinolone) showed the greatest activity (MIC(90), 0.06 µg/ml), with high bactericidal activity and synergy in sitafloxacin-lansoprazole (a proton pump inhibitor) combination. In a Mongolian gerbil model with a H. pylori ATCC 43504 challenge, marked eradication effects were observed at ≥1 mg/kg for sitafloxacin, ≥10 mg/kg for levofloxacin, and ≥10 mg/kg for lansoprazole, reflecting MIC levels for each agent (0.008, 0.25, and 2 µg/ml, respectively). The therapeutic rates were 83.3% for the sitafloxacin (0.3 mg/kg)-lansoprazole (2.5 mg/kg) combination and 0% for either sitafloxacin or lansoprazole alone. The maximum serum concentration (C(max)) of sitafloxacin was 0.080 ± 0.054 µg/ml at 30 min, when orally administered at 1 mg/kg. The simultaneous administration of lansoprazole resulted in no difference. In the resistance development assay, MICs of levofloxacin increased 64- to 256-fold with gyrA mutations (Ala88Pro and Asn87Lys), while MICs of sitafloxacin only up to 16-fold with the Asn87Lys mutation. The data suggest that sitafloxacin exhibited superior anti-H. pylori activity with low rates of resistance development in vitro and that, reflecting high in vitro activities, sitafloxacin-lansoprazole combination exhibited strong therapeutic effects in Mongolian gerbils with a C(max) of sitafloxacin that was 10-fold higher than the MIC value at a 1-mg/kg administration.

Helicobacter pylori resistance to metronidazole and clarithromycin in Ireland.

INTRODUCTION: Helicobacter pylori eradication rates have fallen considerably in recent years. Antibiotic resistance is thought to be rising. OBJECTIVES: To examine the levels of resistance to metronidazole (MTZ) and clarithromycin (CLA) in H. pylori, isolates were taken in a reference centre in Ireland from 2007 to 2008 and were compared to a similar cohort from a study in 1997. METHOD: Antimicrobial susceptibilities were tested by E-test. Frequencies of spontaneous metronidazole and clarithromycin resistance were measured on an agar plate containing the antibiotics at concentrations of 2x and 4x minimum inhibition concentration values. Clinical data were obtained from charts, laboratory and endoscopy reports. RESULTS: Two hundred and twenty-two patients were analyzed, 98 were females. Colonies amenable to culture were grown in 219 patients. Thirty-seven had prior attempts at eradication therapy (all with amoxicillin-CLA-proton pump inhibitor. A total of 31.5% of the patients had strains resistant to MTZ and 13.2% of the patients were noted to have strains resistant to CLA. About 8.6% of the patients had strains resistant to both the agents. CLA resistance was 9.3% in those who had no prior eradication therapy compared with 32.4% of those who had. CLA resistance increased from 3.9%, among treatment-naive patients in 1997, to 9.3% in our study. MTZ resistance was 29.1% in the treatment-naive population. In 1997, MTZ resistance in the treatment-naive cohort was 27.1%. MTZ resistance was more likely to occur in females (35.4 vs. 28.5%) than in males. CONCLUSION: This study shows that resistance to CLA among Irish patients infected with H. pylori has increased since 1997. The future of treatment may well lie in the widespread use of sensitivity testing before the treatment. This would promote an accurate treatment.

Structural analysis of the full-length gene encoding a fibronectin-binding-like protein (CadF) and its adjacent genetic loci within Campylobacter lari.

BACKGROUND: The combined sequences encoding a partial and putative rpsI open reading frame (ORF), non-coding (NC) region, a putative ORF for the Campylobacter adhesin to fibronectin-like protein (cadF), a putative Cla_0387 ORF, NC region and a partial and putative Cla_0388 ORF, were identified in 16 Campylobacter lari isolates, using two novel degenerate primer pairs. Probable consensus sequence at the -35 and -10 regions were identified in all C. lari isolates, as a promoter. RESULTS: Thus, cadF (-like) gene is highly conserved among C. lari organisms. Transcription of the cadF (-like) gene in C. lari cells in vivo was also confirmed and the transcription initiation site was determined. A peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins was completely conserved amongst the putative cadF (-like) ORFs from the C. lari isolates. CONCLUSION: The putative cadF (-like) ORFs from all C. lari isolates were nine amino acid larger than those from C. jejuni, and showed amino acid residues 137 -140 of FALG (50% identity), instead of the FRLS residues of the maximal fibronectin-binding activity site demonstrated within C. jejuni CadF. A neighbor joining tree constructed based on cadF (-like) gene sequence information formed a major cluster consisting of C. lari isolates, separating from the other three thermophilic campylobacters.

Demonstration of the absence of intervening sequences within 23S rRNA genes from Campylobacter lari.

Cloning, sequencing and characterization of nearly full-length 23S rRNA genes in 12 urease-positive thermophilic Campylobacter (UPTC) isolates were carried out using two novel PCR primer pairs. Nucleotide sequences of the 23S rRNA genes from the 12 isolates were first shown not to carry any intervening sequences (IVSs) in both the 25 and 45 helix regions. Then, two PCR primer sets were designed in silico for amplification of the helix 25 and 45 regions within 23S rRNA gene sequences from Campylobacter lari. No IVSs were identified within the 23S rRNA genes among a total of 53 isolates of C. lari, following PCR amplification, TA cloning and sequencing procedures. Intact 23S rRNA was identified in all 65 C. lari isolates, resulting in no production of the fragmented 23S rRNA. These data suggest that C. lari may not have any opportunity to interact with any other source of IVSs until now, or has been unable to integrate IVSs into their own genomes.

Improving compliance with helicobacter pylori eradication therapy: when and how?

Compliance with therapy is the single most important factor in Helicobacter pylori (H. pylori) eradication. Poorer levels of compliance with therapy are associated with significantly lower levels of eradication. Numerous factors can contribute to achieving good levels of compliance. These include the complexity and duration of treatment. It is also important that the physician is motivated to ensure eradication is confirmed and the patient is sufficiently informed to empower him or her to achieve high levels of compliance. Compliance is also contingent on medication regimes that are simple, safe, tolerable and efficacious. The opportunity to improve compliance exists at every point of contact between the patient and the medical services. Experts and opinion leaders in the field can play a role by ensuring that physicians are educated and motivated enough to encourage and support compliance with H. pylori eradication therapy. Both patients and physicians need to be aware of the importance of the bacterium in causing disease. The importance of the doctor-patient relationship is paramount. Pragmatic strategies that may be of assistance may come in the form of polypills, combined Blister Packs, adjuvant therapies and modified release compounds. Colleagues such as pharmacists and nurse specialists can also play an important role and should be actively engaged. Structured aftercare and follow up offers the best chance for ensuring compliance and subsequent eradication of the H. pylori pathogen.

Intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella asinigenitalis UCD-1(T) strain.

PCR was performed with Taylorella asinigenitalis UCD-1(T) using two primer pairs constructed in silico for the amplification of the intervening sequences (IVSs) in the first quarter and central regions of the 23S rRNA gene. Following TA cloning and sequencing, the strain was identified to carry heterogeneous and multiple IVSs. Two similar tandem repeat units of 25 and 24 base pairs (bp) with unknown function(s) were identified within the two IVSs in the central region. Secondary structure models of IVSs, containing stem and loop structures, were demonstrated. Although 16S rRNA and 4-5S RNA species were identified in the purified RNA fraction, no 23S rRNAs were evident, resulting in the occurrence of some smaller RNA fragments from approximately 500 to 1,600 bp, in length. Thus, the 23S rRNA primary transcripts may be cleaved into some smaller fragments and IVSs. No IVS transcript was detected by northern blot hybridization analysis. The present and previous results strongly demonstrate the occurrence of heterogeneous and multiple IVSs in 23S rRNA gene sequences and 23S rRNA fragmentation, in T. asinigenitalis.

Molecular nature of methicillin-resistant Staphylococcus aureus derived from explosive nosocomial outbreaks of the 1980s in Japan.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) with Panton-Valentine leukocidin (PVL) genes is increasing worldwide. Nosocomial outbreak-derived (hospital-acquired) MRSA (HA-MRSA) in Japan in the 1980s was also largely PVL(+). PVL(+) HA-MRSA and CA-MRSA shared the same multi-locus sequence type (ST30) and methicillin resistance cassette (SCCmecIV), but were divergent in oxacillin resistance, spa typing, PFGE analysis or clfA gene analysis. PVL(+) HA-MRSA, which probably originated in PVL(+)S. aureus ST30, was highly adhesive (carrying cna and bbp genes), highly-toxic (carrying luk(PV) and sea genes) and highly drug-resistant. PVL(+) HA-MRSA was once replaced by other PVL(-) HA-MRSA (e.g., ST5), and is re-emerging as CA-MRSA.

Inhibitory effect of antimicrobial agents and anisodamine on the staphylococcal superantigenic toxin-induced overproduction of proinflammatory cytokines by human peripheral blood mononuclear cells.

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), produces superantigenictoxins, such as toxic shock syndrome toxin-1 (TSST-1). TSST-1 abnormally activates T cells to overproduce inflammatory cytokines (such as tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) leading to shock. In this study, we investigated the inhibitory effect of antimicrobial agents and anisodamine (a Chinese herbal extract) on TSST-1-induced cytokine production. Among the macrolides and related agents examined, azithromycin and rokitamycin showed the greatest inhibitory activity against the TSST-1-induced cytokine production. This inhibitory effect was similar to that of anisodamine, which, however, had no inhibitory activity against bacterial growth. Vancomycin, teicoplanin, arbekacin, and linezolid (anti-MRSA and related agents) had no significant inhibitory effect on cytokine production. The inhibitory effect of the drugs on cell proliferation was not significant. These data indicate that some antimicrobial agents, e.g., azithromycin and rokitamycin, manifest anti-superantigenic toxin activity through the inhibition of cytokine production, just like anisodamine.

A Panton-Valentine leucocidin (PVL)-positive community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain, another such strain carrying a multiple-drug resistance plasmid, and other more-typical PVL-negative MRSA strains found in Japan.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was collected from children with bullous impetigo in 2003 and 2004. One strain collected in 2003 was Panton-Valentine leucocidin (PVL) positive. In 2004, a multiple-drug-resistant PVL(+) CA-MRSA strain was isolated from an athlete with a cutaneous abscess. These strains were analyzed by multilocus sequence typing, spa typing, agr typing, coagulase typing, staphylococcal cassette chromosome mec (SCCmec) typing, PCR assay for 30 virulence genes, drug susceptibility testing, pulsed-field gel electrophoresis, and for plasmids. The two Japanese PVL(+) CA-MRSA strains belonged to the globally extant ("pandemic") sequence type 30 (ST30) with SCCmec IV. A transmissible, multiple-drug resistance plasmid emerged in such ST30 strains. The PVL(-) CA-MRSA strains ("domestic" CA-MRSA) accumulated for bullous impetigo, exhibiting new genotypes. Hospital-acquired MRSA of ST91 (but not pandemic ST5) shared common features with the PVL(-) CA-MRSA strain.

Rapid and quantitative detection of blood Serratia marcescens by a real-time PCR assay: its clinical application and evaluation in a mouse infection model.

Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20-60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 10(2)CFU ml(-1)), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 10(7) to 10(8)CFU ml(-1) at a fatal stage (resulting in 100% death), 10(4)-10(5)CFU ml(-1) at a moderately fatal stage (resulting in 50% or more death), and <10(3)CFU ml(-1) at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.

Specific inhibitory action of anisodamine against a staphylococcal superantigenic toxin, toxic shock syndrome toxin 1 (TSST-1), leading to down-regulation of cytokine production and blocking of TSST-1 toxicity in mice.

Toxic shock syndrome toxin 1 (TSST-1), produced by Staphylococcus aureus (including methicillin-resistant S. aureus), is a superantigenic toxin responsible for toxic shock syndrome as well as neonatal TSS-like exanthematous disease. TSST-1 exhibits its deleterious effects by leading to the abnormal proliferation of, e.g., Vbeta2+ T cells and overproduction of proinflammatory cytokines. In the present study we examined the inhibitory effect of a Chinese herbal extract, anisodamine, on TSST-1 using human peripheral blood mononuclear cells (PBMCs). Anisodamine inhibited the production of proinflammatory cytokines better than interleukin-10 (an anti-inflammatory cytokine). The inhibitory effect of anisodamine was greater than that of any tropane alkaloid examined. Anisodamine acted directly on both monocytes and T cells in human PBMCs, and the effect was confirmed at the transcriptional level. Inhibition of NF-kappaB activation was also demonstrated. In contrast, no significant inhibition of Vbeta2+ T-cell proliferation was observed. In mice injected with TSST-1, anisodamine treatment significantly decreased serum proinflammatory cytokine levels and prevented TSST-1-induced death. These results suggest that anisodamine specifically acts against the production of cytokines (inflammatory cytokines in particular) and not against Vbeta2+ T-cell proliferation and that anisodamine may have a beneficial effect on TSST-1-associated disease.

Gene sequences and specific detection for Panton-Valentine leukocidin.

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 x 10(1) CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.

Intrafamilial spread of the same clarithromycin-resistant Helicobacter pylori infection confirmed by molecular analysis.

Clarithromycin-resistant Helicobacter pylori (CRHP) is increasing worldwide, especially in children. We report a family case in which both the mother and child were infected with CRHP. DNA analysis revealed that all of the mother's and daughter's isolates were indistinguishable, suggesting that the same CRHP strain spread between the family members. The spread of CRHP within families may be increasing.