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We reported the case of a patient with leukemia who developed febrile neutropenia after hematopoietic stem cell transplantation. Blood culture results revealed the presence of Streptococcus oralis, while antimicrobial susceptibility testing showed the resistance to penicillin and cephem. Furthermore, isolates were not susceptible to either meropenem or daptomycin but not to vancomycin. S oralis is known to belong to Streptococcus mitis group and be a causative agent of bacteremia in the neutropenic patients, but multidrug resistance of S oralis is rare. Our findings suggest that we might pay attention to the emergence of the microorganisms acquiring multidrug resistance in neutropenic patients., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS ( S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus ) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes ( n = 514), clinical isolates ( n = 31), and reference strains ( n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates ( S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains ( S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris , and S. sinensis . The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys., (Copyright © 2020 Imai, Nemoto, Kodana, Tarumoto, Sakai, Kawamura, Ikebuchi, Mitsutake, Murakami, Maesaki, Fujiwara, Hayakawa, Hoshino, Seki and Maeda.)

Background: Klebsiella variicola and K. quasipneumoniae are new species distinguishable from K. pneumoniae but they are often misidentified as K. pneumoniae in clinical settings. Several reports have demonstrated the possibility that the virulence factors and clinical features differ among these three phylogroups. In this study, we aimed to clarify whether there were differences in clinical and bacterial features between the three phylogroups isolated from patients with bloodstream infections (BSIs) in Japan., Methods: Isolates from all patients with BSIs caused by K. pneumoniae admitted to two hospitals between 2014 and 2017 (n = 119) were included in the study. Bacterial species were identified via sequence analysis, and their virulence factors and serotypes were analyzed via multiplex PCR results. Clinical data were retrieved from medical records., Results: Of the 119 isolates, 21 (17.7%) were identified as K. variicola and 11 (9.2%) as K. quasipneumoniae; K1 serotype was found in 16 (13.4%), and K2 serotype in 13 (10.9%). Significant differences in the prevalence of rmpA, iutA, ybtS, entB and kfu (p < 0.001), and allS genes (p < 0.05) were found between the three phylogroups. However, there were no significant differences in clinical features, including the 30-day mortality rate, between the three organisms, although K. variicola was more frequently detected in patients over 80 years old compared with other Klebsiella species (p < 0.005), and K. quasipneumoniae more frequently occurred in patients with malignancy (p < 0.05)., Conclusions: Our findings demonstrated the differences in bacterial pathogenicity and clinical features among these three phylogroups. Further epidemiological studies into BSI caused by Klebsiella species are warranted.

Purpose: The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles., Methodology: A total of 38 positive BC bottles were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. DNA was extracted from each aliquot using an extraction protocol that combined glass bead beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole-genome sequencing was performed on up to 12 samples through a single MinION flow cell., Results: All GN bacteria identifications made by MinION sequencing for 30 min using the What's In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum β-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes., Conclusion: We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.

We report the case of a patient with advanced gastric cancer and multiple liver metastases, who presented with bacteremia and hepatic gas gangrene caused by Clostridium novyi (C. novyi). The gas gangrene caused abscesses to form within metastatic lesions. This case highlights the antitumor effects of C. novyi in human., (Copyright © 2019. Published by Elsevier Ltd.)

Introduction: Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer. Methods: Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D 2 sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer. Results: Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D 2 reads. Conclusion: We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses.

Purpose: Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles., Methodology: 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION., Results: The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The 'pass' reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D 2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing., Conclusion: We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.

The isolation of a carbapenem-resistant Enterobacter cloacae strain harboring the IMI-1 variant of bla IMI-1 carbapenemase points to the worldwide emergence of multidrug resistant bacteria as a potential source of health care infections. In this report, we describe the first isolation of E. cloacae with bla IMI-1 carbapenemase isolated from a Japanese patient in September 2016. The isolate was resistant to carbapenems, levofloxacin, and aminoglycosides, and heteroresistant to colistin but sensitive to fourth-generation cephalosporins. All microbiology laboratories worldwide should be made aware of these bla IMI-1 -producing subtypes with unusual antibiotic susceptibility profiles., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Inhibition of the enzymatic activity of histone deacetylase (HDAC) is a promising therapeutic strategy for cancer treatment and several distinct small molecule histone deacetylase inhibitors (HDACi) have been reported. We have previously identified a new class of non-peptide macrocyclic HDACi derived from 14- and 15-membered macrolide skeletons. In these HDACi, the macrocyclic ring is linked to the zinc chelating hydroxamate moiety through a para-substituted aryl-triazole cap group. To further delineate the depth of the SAR of this class of HDACi, we have synthesized series of analogous compounds and investigated the influence of various substitution patterns on their HDAC inhibitory, anti-proliferative and anti-inflammatory activities. We identified compounds 25b and 38f with robust anti-proliferative activities and compound 26f (IC50 47.2 nM) with superior anti-inflammatory (IC50 88 nM) activity relative to SAHA., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The benefit of early surgery for IE is yet to be determined in non-Western countries. We conducted this study to evaluate the role of early surgery in infective endocarditis (IE) in Japan., Methods: IE admissions in Japan were prospectively registered using a nation-wide WEB-based registration system (CArdiac Disease REgistration, CADRE). The impact of early surgery on in-hospital mortality was assessed in native valve endocarditis (NVE) and prosthetic valve endocarditis (PVE). Risk factors for in-hospital death were assessed by multiple logistic regression analysis. The propensity score for early surgery was calculated to adjust the impact of early surgery., Results: From September 2006 to May 2009, 348 NVE and 81 PVE were registered. In NVE, early surgery was preferable in every quartile stratified with the propensity score and the summary odds ratio (OR) and 95% confidence interval (CI) was 0.12 (0.05-0.31). The predictors of in-hospital death were Staphylococcus aureus infection (OR 3.5, 95% CI 1.26-9.7), heart failure (OR 6.74, 95% CI 2.43-18.7) and early surgery (OR 0.07, 95% CI 0.03-0.2). In PVE, the predictors of in-hospital death were age (OR 1.09, 95% CI 1.01-1.18), S. aureus infection (OR 5.8, 95% CI 1.4-24.01) and heart failure (OR 7.44, 95% CI 1.81-30.67), whereas early surgery was not (OR 0.51, 95% CI 0.12-2.16)., Conclusion: Early surgery for NVE is associated with improved survival in a wide range of clinical subgroups in Japan. In PVE a survival benefit of early surgery is not clear., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Among five American Type Culture Collection (ATCC) Citrobacter strains, qnrB60 in Citrobacter freundii ATCC 6879, an isolate from the preantibiotic era, and qnrB61 in Citrobacter braakii ATCC 51113(T), a type strain belonging to the C. freundii complex, were identified. Meanwhile, a truncated qnrB-like pseudogene was identified in C. freundii ATCC 8090(T) and ATCC 43864. No qnrB-like sequence was found in Citrobacter koseri ATCC 27028(T). These findings underscore the close relationship between this species and qnrB.

Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53(-/-) cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

Background:  It is important to manage a potentially fatal disease such as infective endocarditis (IE) based on evidence and guidelines for treatment published by academic societies. To clarify the current status of IE in Japan, we conducted a nationwide survey of IE (CArdiac Disease REgistration-Infective Endocarditis [CADRE-IE])., Methods and Results:  We collected data on every aspect of IE using web-based survey. Only a Japanese Circulation Society-certified cardiologists who had managed a case of IE could register the patient when the outcome was known. There were 513 cases (320 men, 193 women) from 114 institutions. The median age was 61 years and more than 80% of the patients had underlying cardiac diseases. The majority was valvular heart disease, in which mitral regurgitation was a leading cause. Most of the patients had some predisposing factors, among which, decayed teeth or periodontitis was a leading factor. Streptococci were found in approximately 52% and staphylococci were found in 32% in positive-culture cases. Methicillin-resistant Staphylococcus aureus was found in 7.5%. Congestive heart failure was found in 43% and intracranial complications were found in 31%. Most of the patients were discharged (65%) but in-hospital death occurred in 11%., Conclusions:  IE is still a lethal disease affecting older patients. The information obtained from the survey should be very helpful for physicians.

Infective endocarditis (IE) is traditionally diagnosed by microbiological analysis of blood cultures, following which therapeutic antibiotics are chosen based on antimicrobial sensitivity tests. However, such conventional techniques do not always lead to an accurate etiological diagnosis. Recently, PCR analysis of the 16S rRNA gene has been employed to identify organisms isolated from excised heart valves. In this study, we analyzed 19 valve samples from patients with confirmed IE, as identified by Duke's criteria. Using broad-range PCR amplification, followed by direct gene sequencing, pathological agents were identified in all samples. Although blood cultures yielded negative results in 4 cases, PCR analysis of valve samples showed positive identification of causative organisms. In 3 cases, there was a difference between blood culture and PCR in identification of pathological agents, which are likely to be misidentified by the conventional method based on the phenotypic database. Postoperative antibiotics were chosen considering the severity of lesions and the results of PCR, Gram staining, and valve cultures. All patients were cured without relapse. The broad-range PCR method was therefore beneficial for the management of IE because it enabled us to identify pathogens directly from the site of infection, even organisms that were difficult to culture or likely to be misidentified by the conventional culture method. Identification of the agents provided precise knowledge of the microbiological spectrum involved in the cases of IE.

Clinical results for linezolid (LZD) treatment of hospital-acquired pneumonia (HAP) caused by methicillin-resistant Staphylococcus aureus (MRSA), particularly microbiologically evaluable or severe cases, are limited in Japan. A prospective observational study was conducted in order to assess the usefulness of LZD in Japanese patients with MRSA pneumonia. The study tracked fifteen participants treated with LZD for pneumonia who met the criteria of the HAP guidelines and were confirmed to have pneumonia caused by MRSA. Of these, six were severe and 13 had received antibiotic treatment before treatment with LZD. Of the 13 participants assessed for their clinical responses, seven were rated as cures, three were rated as failures, and three were indeterminate. The overall cure rate (cure/cure + failure) was 70.0% (7/10), and the cure rate by severity was 33.3% (1/3) for severe cases and 85.5% (6/7) for moderate cases. The one severe case with a clinical response rating of cure had failed to respond to vancomycin. Among the seven participants with a clinical response rating of cure, the microbiological response was eradication in three, presumed eradication in three, and indeterminate in one. Three serious adverse events occurred in two of the 15 participants, but none were considered to be causally related to LZD. The results suggest that LZD has high potential for severe and multidrug-resistant cases. A higher cure rate was achieved in moderate cases. In cases of pneumonia that are most likely MRSA infections with poor prognosis, it was suggested to be important for patient outcome to implement the most effective therapy before the patient's condition becomes serious.

Aerococcus urinae is a endocarditis rare causative organism with low virulene. We report an A. urinae endocarditis case treated by aortic valve replacement. An 80-year-old woman hospitalized for urinary tract infection and hydronephrosis due to three-week renal calculi. Blood culture on admission isolated Streptococcus acidominimus. During the course, she was transferred to our care for surgical intervention after developing congestive heart failure due to severe aortic regurgitation. Echocardiographic findings indicated infective endocarditis. She underwent aortic valve replacement, and gram staining of the resected valve tissue showed gram-positive cocci, although valve culture was negative. PCR amplification and DNA sequencing using the valve material matched an A. urinae sequence. The woman recovered and was discharged six weeks after antibiotic treatment.

The innate immune system of humans recognizes the human pathogenic fungus Candida albicans via sugar polymers present in the cell wall, such as mannan and beta-glucan. Here, we examined whether nucleic acids from C. albicans activate dendritic cells. C. albicans DNA induced interleukin-12p40 (IL-12p40) production and CD40 expression by murine bone marrow-derived myeloid dendritic cells (BM-DCs) in a dose-dependent manner. BM-DCs that lacked Toll-like receptor 4 (TLR4), TLR2, and dectin-1, which are pattern recognition receptors for fungal cell wall components, produced IL-12p40 at levels comparable to the levels produced by BM-DCs from wild-type mice, and DNA from a C. albicans pmr1Delta null mutant, which has a gross defect in mannosylation, retained the ability to activate BM-DCs. This stimulatory effect disappeared completely after DNase treatment. In contrast, RNase treatment increased production of the cytokine. A similar reduction in cytokine production was observed when BM-DCs from TLR9(-/-) and MyD88(-/-) mice were used. In a luciferase reporter assay, NF-kappaB activation was detected in TLR9-expressing HEK293T cells stimulated with C. albicans DNA. Confocal microscopic analysis showed similar localization of C. albicans DNA and CpG-oligodeoxynucleotide (CpG-ODN) in BM-DCs. Treatment of C. albicans DNA with methylase did not affect its ability to induce IL-12p40 synthesis, whereas the same treatment completely eliminated the ability of CpG-ODN to induce IL-12p40 synthesis. Finally, impaired clearance of this fungal pathogen was not found in the kidneys of TLR9(-/-) mice. These results suggested that C. albicans DNA activated BM-DCs through a TLR9-mediated signaling pathway using a mechanism independent of the unmethylated CpG motif.

Antimicrobial therapy of Mycobacterium abscessus infection is difficult because there are relatively few effective treatment regimens and single-agent therapy frequently fails clinically. In light of the lack of data on the susceptibility profile of M. abscessus strains recovered from infections in Japan, the in vitro activity of imipenem in combination with clarithromycin, levofloxacin, moxifloxacin, minocycline, amikacin or tobramycin was investigated by the checkerboard method and compared with the combination amikacin and cefoxitin. The combination of imipenem with moxifloxacin, levofloxacin or clarithromycin had a higher synergistic and/or additive effect than amikacin and cefoxitin. A decrease in the MIC(90) value (minimum inhibitory concentration for 90% of the organisms) was observed in the presence of imipenem for clarithromycin, minocycline, levofloxacin and moxifloxacin. The data suggest that a combination regimen including imipenem may be a good choice in empirical treatment of M. abscessus infection.

Solid organ transplantation is becoming increasingly common in Japan. Despite invasive fungal infections being less common than bacterial and viral infections, fungal infections still result in a higher mortality rate. Empiric and pre-emptive therapy plays an important role in management of invasive fungal infections, because successful treatment is difficult after a definite diagnosis of an invasive disease, especially invasive aspergillosis. Given this situation, to improve outcome, high risk patients need to be identified and antifungal prophylaxis is mandatory in preventing the development of the disease. However, antifungal prophylaxis for solid organ transplant recipients remains controversial. New antifungal agents might change the choice of fungal prevention and treatment.

The Japanese Circulation Society appointed a committee to develop guidelines for the prevention, diagnosis and management of infective endocarditis in Japan. In making such guidelines, the committee required information on the current clinical characteristics of infective endocarditis and therefore performed a nationwide questionnaire survey of cases from 2000 and 2001. In total, data were received for 848 cases from 277 of the 817 hospitals surveyed. Mean age was 55+/-18 years and most patients were aged in their 50 s or 60 s; 53.9% of the patients had infective endocarditis of unknown origin (without any prior predisposing conditions or procedures) and the second most common etiology was post dental procedures. The most common microorganism was Gram-positive cocci (345 streptococci and 221 staphylococci) and methicillin resistant Staphylococcus aureus (MRSA) was found in 7.3%. Although more than 90% of cases with Streptococcus viridans were sensitive to penicillin G, 6.6% were resistant. All MRSAs were sensitive to vancomycin. The information obtained from the survey assisted in the making of the guidelines, which should become an indispensable tool for all clinicians.

Invasive fungal infections remain one of the challenging complications after solid organ transplantation. Although, having a lower incidence than viral and bacterial infections, fungal infections carry the risk of higher mortality. Management of fungal infections involves several difficulties: the difficulty of early diagnosis including the lack of reliable diagnostic methods, limited effective therapy associated with side effects and drug interaction with immunosuppressive drugs. Reduction of risk factors and preemptive antifungal therapy prevents and improves the outcome of invasive fungal infections in heart transplant recipients.

Pacemaker infection is one of the severe complication of pacemaker implantation. We report a case of pacemaker infection caused by Staphylococcus schleiferi which is a coagulase-negative staphylococcus, and its relation with human infection is not well characterized. In 1994, a 80-year-old male presented with a pacemaker pocket infection, cutaneous inflammation but no fever 2 months after insertion of a pacemaker. S. schleiferi was isolated from the pus. The patient was given cefazolin for 5 days. One month later he was readmitted because of cutaneous inflammation and the extruded generator was removed. S. schleiferi was isolated from the generator. After the patient was treated with cefazolin for 3 weeks, four consecutive wound cultures were all negative. A new generator was inserted on the same side. One month after re-insertion, the patient again presented a cutaneous inflammation, and S. schleiferi was isolated from the pus as well as the generator and the leads on their removal. Twenty-six days later, a new pacing system was inserted on the other side. There was no further recurrence of the infection. Removal of the entire pacing system was necessary to cure the infection. We expect further information of human infections caused by S. schleiferi.

On the basis of two case reports it is suggested that serious convulsions may occur in patients treated with flucoazole when serum trough concentrations exceed 80 microg/mL. The authors recommend monitoring fluconazole concentrations during high-dose therapy in patients with poor kidney function.

A sixty-four-year-old male patient was admitted on 13 April 1995 with diagnosis of old pulmonary tuberculosis and pulmonary aspergilloma. He developed a tarry stool and frequent loose motion in early November 1995. Histopathological findings of endoscopic biopsy from the duodenum and colon were suggestive of secondary amyloidosis. In spite of antibiotic and steroid pulse, he developed shock, and massive infiltration shadow appeared in chest X-ray. The patient died on 29 December 1995. The postmortem examination in the specimens of the lung, heart, kidney, liver, and spleen revealed hyphae of Aspergillus sp. and in the specimens of the lung, kidney, spleen, esophagus, adrenal gland, and thyroid revealed amyloid. He was finally diagnosed as invasive aspergillosis with secondary amyloidosis.

The deep-seated mycosis occurred in immunocompromized patients. Normally, the deep-seated mycosis became sever infection because of the defects of host defense or the adverse effects of antifungal agents. The major factors of the reason on the severity of the deep-seated mycosis depends on the pathogenesity and the drug resistance for antifungal agents in infected fungi. The clinical factors related with hosts defenses are important to the other reason on the severity. We investigated that the multiple drug resistant (MDR) mechanism may be one of the major roles plays in the azole resistant Candida albicans strains isolated form the patients with oropharyngeal candidiasis infected HIV. We analyzed which clinical factors are related with the prognosis of the patients with pulmonary cryptococcosis and aspergilloma. The titer of cryptococcal capsular antigen was earlier improve in the patient without underline disease than in the patients with underline diseases diagnosed pulmonary cryptococcosis. CRP was higher in the death cases in the patients with pulmonary aspergilloma, compared with alive cases.

Saccharomyces cerevisiae, a yeast of low pathogenic potential, is a rare but well-documented cause of invasive infections in humans. The yeast Candida albicans is a much commoner cause of significant and life-threatening infections. In such infections the heat shock protein hsp90 is an immunodominant antigen associated with protective humoral immunity. In this study it was shown that over-expression of S. cerevisiae hsp90, the amino acid sequence of which shows 84% identity to C. albicans hsp90, significantly increased the virulence of a laboratory strain of S. cerevisiae in mice, both in terms of colony counts in the kidney, liver, and spleen, and in terms of mortality. This is the first direct evidence that hsp90 is a virulence factor.

Basic and clinical evaluation of lysis centrifugation using Isolator 10 was performed and compared with culture bottle methods. Blood, which was inoculated with C. albicans, C. tropicalis or C. parapsilosis, was cultured using lysis centrifugation or culture bottle method. The culture duration of C. albicans and C. parapsilosis by lysis centrifugation was shorter than that by the culture bottle (BHI release). C. albicans, C. parapsilosis and C. tropicalis became culture-positive within 1 or 2 days after culture by lysis centrifugation. C. albicans and C. parapsilosis became positive 4 days and C. tropicalis 6-8 days after culture by the culture bottle (BHI super). Clinical evaluation of both blood culture method as performed from April 1990 to March 1994. Sixty samples (4.4%) were positive out of 1370 samples. Twenty eight samples (7.2%) were positive out of 389 samples, which were examined by both methods. Twenty two samples were positive by both methods and the rest of the 6 samples was positive only by Lysis centrifugation.

The G test containing factor G, fractioned from the Limulus lysate, was used to detect (1-3)-beta-D-glucan in a rat model of aspergillosis. Aspergillus fumigatus strain MF-13, 1 x 10(4) conidia, were inoculated transtracheally into rats treated with cortisone acetate (100 mg/kg) and fed a low-protein (8%) diet. Increased serum (1-3)-beta-D-glucan was found on the sixth day after inoculation in concentrations of 370 +/- 178 pg/ml (mean +/- SD) in untreated controls, and 154 +/- 43 pg/ml in rats treated with 0.5 mg/kg of amphotericin B. On day 11 (1-3)-beta-D-glucan concentrations were 2,590 +/- 2,940 pg/ml and 448 +/- 442 pg/ml, respectively. The elevation in levels of (1-3)-beta-D-glucan increased in correlation with the elevation of galactomannan antigen titers; (1-3)-beta-D-glucan is thus measurable during experimental aspergillosis in rats.

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and > 75 mg/kg in ABLC. In vitro antifungal activities against Candida albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei were evaluated by the agar plate dilution method. The activities were unchanged against C. albicans, but MICs increased more than four fold in 18 of the 20 strains other than C. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected with C. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.

A latex agglutination test for cryptococcal antigen, the Eiken Latex test (Eiken, Tokyo, Japan), was compared with a monoclonal antibody-based agglutination assay, Pastorex Cryptococcus (Diagnostics Pasteur, Marneur-la-Coquette, France). In a murine model of disseminated cryptococcosis, the kinetics of the antigen titers by the Eiken Latex were similar to those by the Pastorex Cryptococcus, but sensitivity was much higher. In HIV-negative patients with pulmonary cryptococcosis, a cryptococcal antigen was detected in 6 of 10 patients by the Eiken Latex test and in only 3 of those patients by the Pastorex Cryptococcus. The results indicate that the Eiken Latex is more sensitive for the detection of the cryptococcal antigen, even in non-disseminated cryptococcosis. The sensitivity and specificity of the Eiken Latex were examined using 195 sera from 25 patients with pulmonary cryptococcosis and 170 patients with non-cryptococcosis. The cutoff value of > or = 1:8 showed a sensitivity of 76% (19/25) and a specificity of 98.9% (168/170).

(1-3)-beta-D-Glucan (beta-glucan) is a major structural component of fungi. The G test is a direct method to detect beta-glucan using fractionated (1-3)-beta-D-glucan-sensitive component, factor G, eluted from the limulus lysate. Previously, we reported that the G test is a more sensitive method than the mannan detection assay for the serological diagnosis of Candida infection. In this study, we discuss beta-glucanemia in patients with pulmonary aspergillosis and cryptococcosis. The concentration of beta-glucan was less than 10 pg/ml in 9 of 10 cases of pulmonary cryptococcosis, except for one case receiving hemodialysis (16.5 pg/ml). beta-Glucan increased in 3 cases of invasive pulmonary aspergillosis (27-937 pg/ml). Galactomannan antigen was positive in all of those cases. In 8 cases of aspergilloma, which showed fungus ball on roentgenogram, the mean concentration of beta-glucan was 67.1 +/- 92.7 pg/ml. Two of 8 cases were positive for galactomannan antigen. One of three cases of PAIC (productive aspergilloma on the inner wall of a cavity) and one case of chronic necrotizing pulmonary aspergillosis showed slightly increased levels of beta-glucan and positive results of galactomannan antigen test.

The efficacy of the combination therapy of fluconazole plus flucytosine against pulmonary cryptococcosis was investigated. The minimal inhibitory concentrations of antifungal agents were measured by growth inhibition on Yeast-morphology agar plates, and the fractional inhibitory concentration index of each antifungal combination were calculated by checker-board titration. Eight of Cryptococcus neoformans clinical isolates showed sensitivity for AMPH, 5-FC, MCZ and ITZ. C. neoformans showed resistance against FLCZ. The combination of FLCZ plus 5-FC showed synergistic effect against C. neoformans. Three cases of pulmonary cryptococcosis were treated by combination therapy of fluconazole plus flucytosine. After treatment, C. neoformans isolates were eradicated and the chest X-ray improved remarkably. The combination therapy of fluconazole plus flucytosine was effective for the treatment of pulmonary cryptococcosis.

A 57-year-old man with pulmonary tuberculosis underwent left upper lobectomy in 1984. In 1987, chest X-ray showed a fungus ball, and Aspergillus species was isolated from sputum. He was treated by intracavitary injection of amphotericin B (AMPH) in June, 1992. However, no change was observed in the chest CT scan after a total dose of 1,000 mg of AMPH. Combination therapy of intravenous administration of 100,000 units of urinastatin and intracavitary injection of AMPH resulted in complete disappearance of the fungus ball on chest CT scan. This report describes case of pulmonary aspergilloma successfully treated with the combination of AMPH and urinastatin.

Liposomal amphotericin B was evaluated for toxicity, pharmacokinetic properties and therapeutic efficacy in invasive pulmonary aspergillosis in Sprague-Dawley rats. The protective effect of liposomes against the toxicity induced by repeated amphotericin B infusions was observed in a survival study of rats, thereby allowing higher dosages to be administered. Pharmacokinetic studies showed that liposomes led to high levels of amphotericin B in the spleen and liver. Drug levels in the lung were higher at ten minutes after intravenous injection of liposomal amphotericin B at 20 mg/kg than of free amphotericin B at 1.5 mg/kg. Efficacy was assessed in rats treated with cortisone acetate, fed a low-protein diet and infected transtracheally with 8 x 10(4) Aspergillus fumigatus spores to cause fetal pneumonia and pulmonary bleeding. Dosages of 20 or 4 mg/kg of liposomal amphotericin B, or 1.5 mg/kg of free amphotericin B were administered intravenously to rats once daily for 8 days. Control rats received saline. Twenty mg/kg of liposomal amphotericin B was as effective on survival rate as 1.5 mg/kg of free amphotericin B. However, 4 mg/kg of liposomal amphotericin B was less effective than 1.5 mg/kg of free amphotericin B, prolonging the mean survival time of rats treated with saline, which showed elevation of galactomannan antigen titers of Aspergillus fumigatus in sera and invasive proliferation or mycelia with bleeding in histopathological sections of the lung. We conclude that liposomal amphotericin B reduces toxicity of the drug, thereby increasing the concentration of the drug in the lung by high-dose administration, but decreases the efficacy of the drug in experimental pulmonary aspergillosis.

The incidence of fungal isolation in 711 clinical specimens from the respiratory tract was investigated in 159 patients with pulmonary diseases. Fungi were isolated from 14/19 pharynx swab samples, 246/342 sputum samples, 34/147 bronchial aspirate samples, 18/97 bronchial lavage samples, 4/17 transbronchial lung biopsy samples, and 5/60 plugged catheter brush samples. Contamination by fungi in normal flora was prevented by using an aseptic procedure for sampling specimens from the respiratory tract. The isolation rate of Candida spp. from sputum was 70.0% (C. albicans: 151 strains, C. glabrata: 43 strains, C. tropicalis: 25 strains, C. lusitaniae: 4 strains, C. krusei: 3 strains, C. guilliermondii: 3 strains and C. paraspilosis: 1 strain). The isolation rates of C. neoformans and A. fumigatus from sputum were 6.4% and 8.8%, respectively. Isolated fungi from bronchial aspirate included 12 strains of C. albicans, 6 strains of C. tropicalis, 5 strains of A. fumigatus and 2 strains of C. neoformans. Isolated fungi from bronchial aspirate revealed 6 strains of C. albicans, 2 strains of C. glabrata, 1 strain of C. neoformans and 6 strains of A. fumigatus. The isolation rates of fungi from sputum obtained from patients with pulmonary aspergilloma and cryptococcosis were 66.5% and 50.0%, respectively. The isolation rate of fungi from sputum of patients with underlying diseases was higher than that of patients with no underlying disease.

The serodiagnostic tests, beta-glucan, mannan, candida antigen by Cand-Tec, and D-arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta-glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test-D and Endospecy), which was more than 60 pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand-Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D-Arabinitol was the least sensitive, because a D-arabinitol/creatinine ratio greater than 2.0 mumol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti-fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.

Factor G, the coagulation enzyme from limulus amebocytes, is activated by (1-->3)-beta-D-glucan but not by endotoxin. The endotoxin-specific limulus test, which is devoid of factor G, reacts only with endotoxin. However, the conventional limulus test which includes factors G and C, reacts with not only endotoxin but also with (1-->3)-beta-D-glucan. In this study, the culture supernatant of Candida albicans in RPMI medium activated the conventional limulus test, but not the endotoxin-specific limulus test. All five rabbits inoculated intravenously with Candida albicans (1.0 x 10(7) CFU/rabbit) showed increased levels of reactivity with the conventional limulus test, whereas no elevation in the levels of the endotoxin-specific limulus test was observed in the sera of any infected rabbits. Only one serum sample from rabbit No. 5 on day 8 showed a positive Cand-tectest. The difference in the titers of the two limulus tests was suggestive of a diagnosis of Candida infection.

Picogram quantifies per ml of endotoxin showed a positive limulus test. The concentration more than ten pg/ml of curdlan ((1-3)-beta-D-glucan) was also positive to a conventional limulus test, which contains a factor G. One nanogram per ml of CM-curdlan was positive, but higher concentration of 10 pg/ml of CM-curdlan decreased its optical density of the conventional limulus test. More than one hundred ng/ml of mannan from Saccharomyces cerevisiae did not react with factor G. However, a culture supernatant of Saccharomyces cerevisiae activated factor G. This result suggested that beta-glucan activated factor G. In addition, culture supernatant of Aspergillus fumigatus, Rhodotorula rubra, Candida parapsilosis, Candida tropicalis, Candida krusei also activated factor G, but Cryptococcus neoformans did not activate it.

A 76-year-old man who was admitted to the hospital because of chronic renal insufficiency and chronic hepatitis died of Corynebacterium group JK pneumonia, after showing a slight improvement by treatment of Staphylococcus aureus with sulbactam/cefoperazone and minocycline. Transtracheal aspiration (TTA) just before his death revealed numerous gram-positive bacilli phagocytized by many neutrophils and more than 10(8) colony forming units (CFU)/ml of Corynebacterium group JK. A drug susceptibility test showed Corynebacterium group JK was resistant to many antibiotics, with the exception of vancomycin and amikacin.

We compared two latex agglutination tests for serum candidal antigen in murine models of systemic infection and gastrointestinal colonization. In the intravascular-infection model, mannan detection test was positive at 2 hours after inoculation, but Cand-Tec was less sensitive than mannan detection test in this model. In the gastrointestinal colonization model followed by systemic infection in neutropenic mice, the mannan antigen test became positive after 3 weeks of colonization with increased number of Candida of the stool, and Cand-Tec became positive after 5 weeks in neutropenic mice.

A retrospective study was done to determine the prevalence of anti-HTLV-I antibodies in patients with pulmonary cryptococcosis. None of the 19 patients with pulmonary cryptococcosis had underlying immunodeficiency. Anti-HTLV-I antibody was present in 6 (32%) of 19 patients with pulmonary cryptococcosis, a significantly higher prevalence than found in patients with bronchial asthma (4 (7%) of 58) (p less than 0.01, chi-square test). No statistical difference was noted when anti-HTLV-I antibody seropositivity was compared to that of patients with pulmonary tuberculosis (16% (17/105)), lung cancer (17% (22/129)) and pneumonia (9% (6/64)). A reduced cellular immunity as shown by lymphopenia, the CD4/CD8 ratio, and purified protein derivative skin test was found in only 1 (5%) of 19, 2 (12%) of 17, and 6 (33%) of 18 patients, respectively. These results do not explain the susceptibility to pulmonary cryptococcosis in HTLV-I carriers. This is the first report of high prevalence of pulmonary cryptococcosis in HTLV-I carriers and it raises the question whether HTLV-I carriers are more susceptible to opportunistic infections and other malignancies probably due to subtle immunological abnormalities.