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Introduction Culture tests are used to diagnose infections, but there are various problems such as low sensitivity in detecting infections in orthopedic cases. To address this problem, next generation sequencing (NGS) analysis, which can comprehensively search for bacterial genes, is being applied clinically. In this study, we examined whether NGS analysis was useful in evaluating infections in orthopedic cases. Methods The participants were 23 patients suspected of having an infection between 2016 and 2017. Samples were collected from tissues suspected of being infected and were subjected to culture tests and NGS analysis, and the positive rates from the culture tests and from the NGS analysis were compared. We also attempted to determine cutoff value for the NGS analysis. Results A total of 20 cases were ultimately diagnosed as infections and 3 cases were diagnosed as non-infections. The sensitivity of the culture tests was 70%, and the sensitivity of the NGS analysis was 55%. When the NGS analysis was performed with the diversity index set to the cut-off value, the sensitivity was 75% for the Simpson index. In this study, the sensitivity was 90% when the analysis was performed using the NGS index, which is a combination of the diversity index and the OTUs (operational taxonomic units) value. Conclusion NGS analysis using the NGS index showed excellent sensitivity and specificity compared to culture tests. NGS analysis is therefore a useful modality for assessing infections in orthopedic cases.

BACKGROUND: Hospital outbreaks of antimicrobial-resistant (AMR) bacteria should be detected and controlled as early as possible. AIM: To develop a framework for automatic detection of AMR outbreaks in hospitals. METHODS: Japan Nosocomial Infections Surveillance (JANIS) is one of the largest national AMR surveillance systems in the world. For this study, all bacterial data in the JANIS database were extracted between 2011 and 2016. WHONET, a free software for the management of microbiology data, and SaTScan, a free cluster detection tool embedded in WHONET, were used to analyse 2015–2016 data of eligible hospitals. Manual evaluation and validation of 10 representative hospitals around Japan were then performed using 2011–2016 data. FINDINGS: Data from 1,031 hospitals were studied; mid-sized (200–499 bed) hospitals accounted for 60%, followed by large (≥ 500 beds) hospitals (24%) and small (< 200 beds) hospitals (16%). Large hospitals resulted in more clusters detected. Most of the clusters included five or fewer patients. From the in-depth analysis of 10 hospitals, approximately 80% of the detected clusters were unrecognised by infection control staff because the bacterial species involved were not included in the priority pathogen list for routine surveillance. In particular, in two hospitals, clusters of more susceptible isolates were detected before outbreaks of more resistant pathogens. CONCLUSION: WHONET-SaTScan can automatically detect clusters of epidemiologically-related patients based on isolate resistance profiles beyond lists of high-priority AMR pathogens. If clusters of more susceptible isolates can be detected, it may allow early intervention in infection control practices before outbreaks of more resistant pathogens occur.

Background The epidermal growth factor receptor (EGFR) is a therapeutic target for patients with non-small cell lung cancer (NSCLC). Cetuximab is an anti-EGFR monoclonal antibody that inhibits EGFR signaling and proliferation of colorectal cancer and head and neck cancers. Since only few NSCLC patients benefit from cetuximab therapy, we evaluated a novel combination treatment using cetuximab and EGFR small interfering RNA (siRNA) to strongly suppress EGFR signaling and searched for a biomarker in NSCLC cell lines harboring wild-type EGFR. Methods Alterations in EGFR and its downstream genes in five NSCLC cell lines (A549, Lu99, 86-2, Sq19 and Ma10) were assessed through sequencing. The protein expression levels of these molecules were assessed through western blotting. The effect of combination treatment was determined through cell proliferation assay, caspase-3/7 assay, invasion assay, and migration assay. Results All cell lines were harboring wild-type EGFR, whereas KRAS, PTEN, TP53 and TP53 were mutated in A549 and Lu99; Lu99 and Sq19; Lu99, 86-2, Sq19 and Ma10; and A549, 86-2, and Sq19 cell lines, respectively. PTEN was not expressed in Sq19, and LKB1 was not expressed in both A549 and Sq19. TP53 was not expressed in both A549 and Lu99. The combination of cetuximab and EGFR siRNA significantly suppressed cell proliferation in 86-2, Sq19 and Ma10, which express wild-type KRAS. It induced apoptosis in A549, 86-2 and Ma10 cells, which express wild type PTEN. The combination treatment had no effect either on cell invasion nor migration in all cell lines. Conclusion EGFR targeted therapy using the combination of cetuximab and EGFR siRNA is effective in NSCLC cell lines harboring wild-type EGFR. Wild-type KRAS may act as a potential biomarker for response to combination treatment by the induction of apoptosis in cells with wild-type PTEN.

Background Peritoneal dialysis (PD)-associated peritonitis caused by nontuberculous Mycobacterium is rare; however, the number of cases has increased over the past decades. Mycobacteroides massiliense is a subspecies of the Mycobacteroides abscessus complex. It has different clinical characteristics compared to the other subspecies of the complex. Previous case reports of PD-associated peritonitis caused by Mycobacteroides abscessus complex have not distinguished the subspecies in detail. Case presentation A 40-year-old man presented with an exit-site and tunnel infection refractory to antibiotic therapy. Peritonitis occurred after simultaneous catheter removal and reinsertion. The Mycobacteroides abscessus complex was detected in the culture of the dialysis effluent. Removal of the PD catheter combined with antibiotics, including macrolides, resulted in a good clinical course. Further analysis of multiplex PCR and the hsp65 gene sequence identified the bacterium as Mycobacteroides massiliense. Conclusions The Mycobacteroides abscessus complex is classified into three subspecies; Mycobacteroides abscessus, Mycobacteroides massiliense, and Mycobacteroides bolletii. These have different characteristics, particularly antibiotic susceptibility. Therefore, clear identification of the subspecies of the Mycobacteroides abscessus complex is necessary for definitive treatment.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from the patients in Japan was conducted by the Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2016. The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period between February 2016 and August 2016 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical Laboratory Standards Institute. Susceptibility testing was evaluated in 1062 strains (143 Staphylococcus aureus, 210 Streptococcus pneumoniae, 17 Streptococcus pyogenes, 248 Haemophilus influenzae, 151 Moraxella catarrhalis, 134 Klebsiella pneumoniae, and 159 Pseudomonas aeruginosa). Ratio of methicillin-resistant S. aureus was 48.3%, and those of penicillin-susceptible S. pneumoniae was 99.5%. Among H. influenzae, 14.1% of them were found to be β-lactamase-producing ampicillin-resistant strains, and 41.1% to be β-lactamase-non-producing ampicillin-resistant strains. Extended spectrum β-lactamase-producing K. pneumoniae and multi-drug resistant P. aeruginosa with metallo β-lactamase were 4.5% and 0.6%, respectively.

ULBP2 is one of the ligands for NKG2D (NKG2DLs). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Previously, we reported high concentrations of serum-soluble ULBP2 (sULBP2) were correlated with poor prognosis in NSCLC patients. As a possible mechanism, we demonstrated that sULBP2 directly suppressed the cytolytic activity of peripheral blood mononuclear cells (PBMCs). However, little is known about the in vivo function of human NKG2DLs on tumor cells. The present study investigates the function of human NKG2DLs in the syngeneic C57BL/6 mice. We generated ULBP2 stably expressing B16 melanoma cell lines (ULBP2-B16). As previously reported, 4-hour 51Cr releasing assay showed NK cell cytotoxicity against the tumor cells was enhanced by ectopic expression of ULBP2 on the tumor cells. IFN-gamma production in NK cells was decreased by non-contact co-culture with ULBP2-B16. To investigate the in vivo function of ULBP2, C57BL/6 mice were inoculated s.c. with mock-B16 cells or ULBP2-B16 cells. ULBP2-B16 tumors grew rapidly compared to mock-B16 tumors. NKG2D blockade by anti-NKG2D antibody injected i.p. suppressed tumor growth of ULBP2-B16 tumors. Spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. NKG2D was expressed on splenic NK cells, while it was rarely expressed on splenic CD8+ T cells. NKG2D was downregulated on NK cells in TILs (NK-TILs) from ULBP2-B16. NKG2D was highly expressed on CD8+ T cells in TILs (CD8+-TILs) from mock-B16 tumors, while its expression was suppressed on these from ULBP2-B16 tumors. Degranulation analysis revealed that ULBP2-B16 reduced activity of NK-TILs and CD8+-TILs compared to mock-B16. In conclusion, ectopic expression of ULBP2 on tumor cells promotes tumor progression via suppression of the activity of NK cells and CD8+ T cells. These findings suggest that neutralization of NKG2DLs may be a promising approach for the treatment of NKG2DLs-expressing tumors. Citation Format: Kosuke Yamaguchi, Naomi Miyake, Yasuhiko Teruya, Kohei Yamane, Naoki Kinoshita, Tomohiro Sakamoto, Masahiro Kodani, Hiroki Chikumi, Akira Yamasaki. Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1770.

Clostridium difficile (C. difficile)-associated diarrhea (CDAD) is a challenging nosocomial infectious disease. C. DIFF Quik Chek Complete assay is widely used to detect glutamate dehydrogenase (GDH) antigen and toxin A/B of C. difficile simultaneously. However, the interpretation of GDH positive/toxin negative results is problematic. We performed a retrospective study of patients with GDH positive/toxin negative results to determine the probability of detecting toxigenic C. difficile and its risk factors. Between April 2012 and March 2017, we investigated cultures of fecal specimens followed by toxin detection tests. The clinical histories of patients with and without toxigenic C. difficile were compared using univariate- and multivariate-analyses. In total, 2675 patients were examined using C. Diff Quik Chek Complete assay. Among 356 GDH positive/toxin negative patients, cultures were performed in 220 cases and toxigenic C. difficile was recovered from 139 (63.2%) specimens. Patients with toxigenic C. difficile had significantly lower body mass index than those without. Over half the GDH positive/toxin negative patients were infected with toxigenic C. difficile. Lower BMI was a CDAD risk factor in this patient population. These data can be utilized to initiate isolation and clinical interventions before confirmatory test results are available. J. Med. Invest. 65:131-135, February, 2018.

We present a case report of a 35-year-old woman who had splenic infarction. She had persistent high fever, systemic joint pain, and abnormal liver function. She was diagnosed with cytomegalovirus and human parvovirus B19 concomitant infection. Her coagulopathy test revealed no abnormal results. She was treated with intravenous ganciclovir for 13 days; consequently, her splenic infarction improved after 7 weeks. As per our knowledge, this is the first case of cytomegalovirus and parvovirus B19 coinfection complicated by splenic infarction. Cytomegalovirus and parvovirus B19 may induce a hypercoagulation state during the acute phase.

Aims Vitamin E is an antioxidant that occurs in 8 different forms (α, β, γ, and δ tocopherol and tocotrienol). Clinical trials of tocopherol supplementation to assess the impact of antioxidant activity in asthma have yielded equivocal results. Tocotrienol exhibits greater antioxidant activity than tocopherol in several biological phenomena in vivo and in vitro. We tested the effect of tocotrienol on human airway smooth muscle (ASM) cell growth and migration, both of which mediate airway remodeling in asthma. Main methods We measured platelet-derived growth factor-BB (PDGF-BB)–induced ASM cell proliferation and migration by colorimetric and Transwell migration assays in the presence and absence of γ-tocotrienol (an isoform of tocotrienol). Key findings PDGF-BB–induced ASM cell proliferation and migration were inhibited by γ-tocotrienol. This effect was associated with inhibition of RhoA activation, but it had no effect on p42/p44 mitogen-activated protein kinase (MAPK) or Akt1 activation. We confirmed that pharmacological inhibition of Rho kinase activity was sufficient to inhibit PDGF-BB–induced ASM cell proliferation and migration. Significance γ-Tocotrienol could impart therapeutic benefits for airway remodeling in asthma by inhibiting human ASM cell proliferation and migration.

Epidermal growth factor receptor (EGFR) gene mutation testing is essential for choosing appropriate treatment options in patients with advanced non-small cell lung cancer (NSCLC). However, a time delay occurs between histological diagnosis and molecular diagnosis in clinical situations. To minimize this delay, we developed a novel point-of-care test for EGFR mutations, based on a high-speed real-time polymerase chain reaction (PCR) system designated here as ultrarapid PCR combined with highly accurate bronchoscopic sampling. We investigated whether our system for detecting EGFR mutations was valid by comparing test results with those obtained using a commercialized EGFR mutation test. We obtained small amounts of bronchial lavage fluids after transbronchial biopsies (TBBs) were performed on enrolled patients (n=168) who underwent endobronchial ultrasonography using a guide sheath (EBUS-GS). EGFR mutation analysis was performed by ultrarapid PCR immediately after EBUS-GS-TBBs were obtained (on the same day). After pathological diagnoses of NSCLC, EGFR mutation status in formalin-fixed, paraffin- embedded samples was confirmed by the PCR-invader method, and the concordance rates between the PCR methods were compared. The total diagnostic yield of EBUS-GS-TBB was 91.0%. The positive concordance rates for detecting 19del and L858R with the ultrarapid PCR and PCR-invader methods were both 100%. Negative concordance rates were 97.2 and 98.1%, respectively. We also demonstrated a dramatic effect of early erlotinib administration, based on ultrarapid PCR results, for a 52-year-old woman suffering from respiratory failure due to severe intrapulmonary metastases with poor performance status. In conclusion, ultrarapid PCR combined with EBUS-GS-TBB enabled rapid and reliable point-of-care testing for EGFR mutations.

Mutations in the epidermal growth factor receptor (EGFR) gene are associated with a favorable clinical response to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib in non-small cell lung cancer (NSCLC). We present here, a new method for the rapid detection of the two most common EGFR mutations (delE746-A750 and L858R) from clinical samples. The methodology involves the combination of newly designed mutation-specific primers and a novel real-time PCR machine with an innovative thermo-control mechanism that enables ultrarapid PCR. We evaluated this method using a cell mixture composed of various ratios of lung cancer cells harboring mutated or wild-type EGFR, lung cancer tissues obtained by surgery, and a cytology sample obtained by bronchoscopy from a lung cancer patient. In the cell mixture analysis, our method detected 0.1% of cells with delE746-A750 and 1% of cells with L858R among cells with wild-type EGFR. In 143 lung cancer tissues, the result of this assay was concordant with those of direct sequencing in 138 samples. The five samples with discordant results were tested using a PCR-Invader assay and the result matched those of our method at 100%. We also successfully detected EGFR mutations in the lavage obtained from a lung cancer patient. The turnaround time for this method was

Objective: To define the molecular epidemiology of respiratory viral infections in adult patients. Methods: Nasal and throat swabs were collected from all adult patients with influenza-like illness (ILI), acute respiratory infection (ARI), or severe ARI (SARI) admitted to a tertiary hospital in Surakarta, Indonesia, between March 2010 and April 2011 and analyzed for 19 respiratory viruses and for torque teno virus (TTV) and human gyrovirus (HGyV). Results: Respiratory viruses were detected in 61.3% of the subjects, most of whom had ARI (90.8%, OR = 11.39), were hospitalized (96.9%, OR = 22.31), had asthma exacerbation (90.9%, OR = 8.67), and/or had pneumonia (80%, OR = 4.0). Human rhinovirus (HRV) A43 predominated. Influenza A H3N2, human metapneumovirus (HMPV) subtypes A1 and A2, the influenza B virus, human adenovirus B, and human coronavirus OC43 were also detected. All respiratory viruses were detected in the transition month between the rainy and dry seasons. No mixed respiratory virus infection was found. Coinfections of the influenza A H3N2 virus with TTV, HMPV with TTV, HRV with TTV, and human parainfluenza virus-3 with TTV were found in 4.7, 2.8, 19.8, and 0.9% of the samples, respectively. Conclusions: This study highlights the need to perform routine detection of respiratory viruses in adults hospitalized with ARI, asthma exacerbation, and/or pneumonia.

We herein discovered a highly resistant clinical isolate of Pseudomonas aeruginosa with MICs to amikacin, gentamicin, and arbekacin of 128 μg/mL or higher in a drug sensitivity survey of 92 strains isolated from the specimens of Yoka hospital patients between January 2009 and October 2010, and Achromobacter xylosoxidans was separated from this P. aeruginosa isolate. The sensitivity of this bacterium to 29 antibiotics was investigated. The MICs of this A. xylosoxidans strain to 9 aminoglycoside antibiotics were: amikacin, gentamicin, arbekacin, streptomycin, kanamycin, neomycin, and spectinomycin, 1,024 μg/mL or ≥ 1,024 μg/mL; netilmicin, 512 μg/mL; and tobramycin, 256 μg/mL. This strain was also resistant to dibekacin. This aminoglycoside antibiotic resistant phenotype is very rare, and we are the first report the emergence of A. xylosoxidans with this characteristic.

A 75-year-old woman with aplastic anemia was admitted to our university hospital because of a dry cough that had persisted for a month. Chest computed tomography showed a mass shadow with a central low attenuation area in the lower lobe of the left lung. Filamentous fungus resembling Aspergillus fumigatus was cultured from the specimens obtained by transthoracic needle aspiration biopsy and bronchoalveolar lavage. The initial diagnosis was a lung abscess due to A. fumigatus, although the patient did not respond well to antifungal agents. Subsequently, the filamentous fungus was identified as Aspergillus viridinutans by sequence analysis of the β-tubulin gene, and the patient was successfully treated with combination therapy along with granulocyte colony-stimulating factor. The incidence of A. viridinutans infection is very rare. A. viridinutans is morphologically similar to A. fumigatus; however, the response to antifungal agents is generally worse than that observed in A. fumigatus infections. Therefore, the selection of agents and supplemental therapy is of vital importance in cases of A. viridinutans infection.