Shigefumi Maesaki

Antimicrobial susceptibility and molecular characteristics of 857 methicillin-resistant Staphylococcus aureus isolates from 16 medical centers in Japan (2008–2009): nationwide survey of community-acquired and nosocomial MRSA

This study is a nationwide survey of all clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, including community-acquired MRSA (CA-MRSA), in Japan. A total of 857 MRSA clinical isolates were collected from the 16 institutions throughout Japan that participated in the survey (2008-2009). The drug susceptibility and staphylococcal cassette chromosome mec (SCCmec) typing and the presence of specific pathogenic genes were evaluated. The isolates comprised SCCmec type II (73.6%), type IV (20%), and type I (6%). The percentage of SCCmec type IV isolates was significantly higher in outpatients than in inpatients. Most of the isolated strains were sensitive to vancomycin (VCM, MIC ≤2 μg/mL), linezolid (MIC ≤4 μg/mL), and teicoplanin (MIC ≤8 μg/mL). Although most strains were sensitive to VCM, the MIC value of VCM for SCCmec type II strains was higher than that for SCCmec type IV strains. Only 4 (2.3%) of 171 SCCmec type IV strains were Panton-Valentine leukocidin (lukS/F-PV)-positive. Thus, this result indicates a unique feature of SCCmec type IV strains in Japan. The information in this study not only is important in terms of local public health but will also contribute to an understanding of epidemic clones of CA-MRSA.

Efficacy and safety of micafungin, an echinocandin antifungal agent, on invasive fungal infections in patients with hematological disorders.

The study was conducted as a prospective multicenter trial to evaluate the efficacy and safety of micafungin in patients with invasive fungal infections (IFIs) in hematological disorders. A total of 277 patients was registered, and 197 were assessed for clinical efficacy. The mean dosage and duration of micafungin were 170.7 mg/day and 22.0 days, respectively. The efficacy rates were 87.5% (7/8) for patients with candidiasis, 44.7% (17/38) for probable IFIs, 61.9% (39/63) for possible IFIs and 80.7% (71/88) for those who failed to respond to antibacterials. In patients with febrile neutropenia (below 500/µL), despite broad-spectrum antibacterial treatment over 2 days, 86.3% (44/51) of patients had a favourable response to micafungin. The incidence of adverse events related to micafungin was 14.1% (39/277), but most of them were mild and reversible. These data indicate the usefulness of micafungin as a novel therapeutic drug for both empirical and targeted therapy for IFIs.

Clinical features of pulmonary cryptococcosis in non-HIV patients in Japan

OBJECTIVE To clarify the clinical features of pulmonary cryptococcosis in Japanese non-HIV population. METHODS Retrospective investigation of 151 pulmonary cryptococcosis cases between 1977 and 2012 was executed. The underlying disease (UDs), aggravating factors, radiological characteristics, and treatment were examined. RESULTS Sixty-seven patients (44.4%) had no UDs. The common UDs were diabetes (32.1%) followed by hematologic disease (22.6%), and collagen disease (22.6%). Peripherally distributed pulmonary nodules/masses were most commonly seen. Lesions in the right middle lobe (p = 0.01) and air bronchogram (P = 0.05) were significantly more frequent, respectively, in patients with UDs than patients without them. Azoles were mainly selected for the patients without meningoencephalitis. Mean treatment duration for patients with and without UDs was 6.64 and 2.87 months, respectively. Patients whose pulmonary nodules improved after treatment continued to experience gradual reduction of cryptococcosis antigen titers, even if antigen titers were positive at the time of treatment cessation. The average time for antigen titers to become negative after treatment cessation was 13.1 and 10.7 months for patients with and without UDs, respectively. When groups were compared according to the presence of meningoencephalitis complications, deaths, and survivals, factors contributing to cryptococcosis prognosis included higher age, hypoproteinemia, hypoalbuminemia, steroid use, high C-reactive protein levels, and meningoencephalitis complications. CONCLUSIONS It is crucial to consider the presence of UDs and meningoencephalitis for the choice of antifungals and treatment duration for cryptococcosis in non-HIV patients. Three- and six months-administration of azoles for pulmonary cryptococcosis with or without UDs, respectively is reasonable.

Exacerbation of Invasive Candida albicans Infection by Commensal Bacteria or a Glycolipid Through IFN-γ Produced in Part by iNKT Cells

BACKGROUND The commensal yeast Candida albicans is a major cause of invasive fungal infections. Despite treatment with antifungal agents, the mortality rate attributed to these types of infection is high. Although numerous cases have been reported regarding a poor outcome for patients with bacterial and C. albicans coinfection, the mechanisms by which the coinfecting bacteria exacerbate the C. albicans infection remain elusive. METHODS AND RESULTS We evaluated how glycolipid-mediated activation of invariant natural killer T (iNKT) cells affects the clearance of C. albicans. Surprisingly, C. albicans-infected, glycolipid-treated mice exhibited significantly lower survival rates, increased fungal burden, and higher interleukin (IL)-6 production in the kidneys compared with control mice. Glycolipid-induced exacerbation of C. albicans infection was not observed in interferon-gamma knockout (IFN-γKO) mice. In the C. albicans-infected, glycolipid-treated mice, the number of neutrophils in the blood and bone marrow dramatically decreased in an IFN-γ-dependent manner. Furthermore, mice that were coinfected with C. albicans and nonfermentative gram-negative commensal bacteria exhibited increased fungal burden and inflammatory cytokine production in the kidneys that were dependent on IFN-γ and iNKT cells. CONCLUSIONS Our results indicate that coinfecting commensal bacteria exacerbate C. albicans infection through IFN-γ produced, in part, by iNKT cells.

A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer

BackgroundA simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer.MethodsWe generated specific LAMP primers targeting the 18S–rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species.ResultsOur LAMP method allowed amplification of all targeted 18S–rRNA genes of the reference plasmids with detection limits of 10–100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR.ConclusionsOur diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

Bacteriological Monitoring of Water Reservoirs in Oxygen Humidifiers: Safety of Prolonged and Multipatient Use of Prefilled Disposable Oxygen Humidifier Bottles

Bacterial colony counts in water specimens from oxygen humidifiers that used reusable water reservoirs were compared with counts in water specimens from humidifiers that used prefilled disposable reservoir bottles to evaluate the effects of prolonged and multipatient use of humidifiers. Bacteria were detected after 1 week of operation in water specimens collected from many humidifiers with reusable reservoirs, but no bacteria were detected in water specimens from disposable bottles for up to 12 weeks during use of the humidifier by multiple patients.

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.

A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay

Rapid and easy detection of a single nucleotide point mutation of bacterial genes, which is directly linked to drug susceptibility, is essential for the proper use of antimicrobial agents. Here, we established a detection method using a peptide nucleic acid mediated loop-mediated amplification (LAMP) assay for macrolide (ML)-susceptible Mycoplasma pneumoniae. This assay specifically detected the absence of missense mutations encoding the central loop of domain V in the gene encoding 23S rRNA, which can reduce the affinity for MLs and subsequently generate ML-resistant strains of M. pneumoniae. Reactions were performed at 62°C for 60min and targeted gene amplifications were detected by real-time turbidity with a turbidimeter and naked-eye inspection of a color change. The assay had an equivalent detection limit of 100.0fg of DNA with the turbidimeter and showed specificity against 54 types of pathogens, whereas amplification was completely blocked, even at 100.0pg of DNA per reaction, in the presence of point mutations at 2063A and 2064A. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This method would be a simple and rapid protocol for single nucleotide polymorphism genotyping as point-of-care testing technology without amplification of the sequences carrying the point mutations 2063A and 2064A in ML-resistant M. pneumoniae strains.

Identification of Disseminated Cryptococcosis Using MALDI-TOF MS and Clinical Evaluation

Disseminated cryptococcosis is rare but can often become severe with a poor outcome. Given recent reports that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyser is useful for Cryptococcus species identification, it was applied retrospectively to past cases of disseminated cryptococcosis at our hospital over the past 10 years, and their clinical courses were reviewed. For each case, the retained Cryptococcus spp. were used for identification using both MALDI-TOF MS and genetic sequencing, as well as for drug susceptibility testing. A total of eight cases were found. Cryptococcus spp. were found in cerebrospinal fluid in 3 cases and blood in 5 cases; anti-HIV antibody was either negative or untested. MALDI-TOF MS identified Cryptococcus neoformans as the pathogen in all 8 cases, but genetic testing identified one of these as Cryptococcus curvatus. The outcome was death within 30 days in 5 of the total 8 cases and in 2 of the 3 cases in which C. neoformans was detected in the cerebrospinal fluid, despite regimens and dosages that followed IDSA Guidelines in all 3 cases. Drug susceptibility testing showed no drug resistance that would have affected the therapy. In conclusion, the outcomes were very poor in these drug-susceptible cases, despite treatment in full accordance with standard guidelines. This study confirmed the need to develop newer therapies as well as the high capability of MALDI-TOF MS for the identification of C. neoformans. Genetic testing, however, may be necessary if non-neoformans Cryptococcus is suspected.

National Trends in the Distribution of Candida Species Causing Candidemia in Japan from 2003 to 2014: A Report by the Epidemiological Investigation Committee for Human Mycoses in Japan

The Epidemiological Investigation Committee for Human Mycoses in Japan performed a retrospective epidemiological survey of candidemia and causative Candida species. Data from 2003 to 2014 were collected from 10 Japanese university hospitals. A total of 328,318 blood cultures were included. The prevalence of fungi in all cultures and in positive cultures were 0.58±0.09% and 4.46±0.66%, respectively. Among the results that were positive for Candida species (N=1,921), Candida albicans was the most common species (39.5%) and was followed by Candida parapsilosis (23.3%), Candida glabrata (13.2%), Candida tropicalis (7.1%), Candida krusei (3.2%), and others (13.7%). During the last 6 years, the frequency of C. albicans has significantly decreased in Japan, while that of C. glabrata has increased. Additional surveys are needed to continuously monitor the trends in the distribution of candidemia.