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ABSTRACT: Objectives: The dissemination of difficult-to-treat carbapenem-resistant Enterobacterales (CRE) is of great concern. We clarified the risk factors underlying CRE infection mortality in Japan. Methods: We conducted a retrospective, multicentre, observational cohort study of patients with CRE infections at 28 university hospitals from September 2014 to December 2016, using the Japanese National Surveillance criteria. Clinical information, including patient background, type of infection, antibiotic treatment, and treatment outcome, was collected. The carbapenemase genotype was determined using PCR sequencing. Multivariate analysis was performed to identify the risk factors for 28-day mortality. Results: Among the 179 patients enrolled, 65 patients (36.3%) had bloodstream infections, with 37 (20.7%) infections occurring due to carbapenemase-producing Enterobacterales (CPE); all carbapenemases were of IMP-type (IMP-1: 32, IMP-6: 5). Two-thirds of CPE were identified as Enterobacter cloacae complex. Combination therapy was administered only in 46 patients (25.7%), and the 28-day mortality rate was 14.3%. Univariate analysis showed that solid metastatic cancer, Charlson Comorbidity Index ≥3, bloodstream infection, pneumonia, or empyema, central venous catheters, mechanical ventilation, and prior use of quinolones were significant risk factors for mortality. Multivariate analysis revealed that mechanical ventilation (OR: 6.71 [1.42–31.6], P = 0.016), solid metastatic cancers (OR: 5.63 [1.38–23.0], P = 0.016), and bloodstream infections (OR: 3.49 [1.02–12.0], P = 0.046) were independent risk factors for 28-day mortality. Conclusion: The significant risk factors for 28-day mortality in patients with CRE infections in Japan are mechanical ventilation, solid metastatic cancers, and bloodstream infections.

Background Recently developed rapid real-time reverse transcription PCR (RT-PCR) systems adopting microfluidic thermal cycling technology are ideal for point-of-care (POC) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because the RNA extraction step before real-time RT-PCR is rate-limiting, a direct RNA extraction method (direct method) that adopts chemical viral lysis and eliminates RNA purification steps is preferable for rapid real-time RT-PCR. In the direct method, selecting the transport medium is essential because it may be introduced into subsequent real-time RT-PCR steps, but might inhibit PCR. However, the influence of transport medium on the combination of the direct method and rapid realtime RT-PCR has been yet unstudied. In the present study, we examined the influence of various transport mediums when combining the direct method and rapid real-time RT-PCR of GeneSoC® (GeneSoC® RT-PCR), the recently developed compact PCR system that adapts novel microfluidic thermal cycling technology. Methods To explore the influence of the transport medium on the GeneSoC® RT-PCR, the concordance of the RNA extraction and direct method was evaluated in the clinical samples collected in viral transport medium (VTM) or eSwab®. The sensitivity of GeneSoC® RTPCR combined with the direct method was assessed using spiked samples in generic (H2O and PBS) or commercially available transport media (VTM and eSwab®). Analytical sensitivity was examined using clinical specimens collected from the VTM and eSwab®. The inhibitory effect of PCR inhibitors on clinical specimens was assessed using clinical samples diluted 1,000 times. Results While only 1 copy/reaction of RNA was detected in H2O and eSwab® of the spiked samples, a minimum of 5 copies/reaction was detected in PBS (-) and VTM. Among the clinical specimens tested using the direct method, the detection of viral RNA was unstable in the samples containing less than 100 copies/reaction viral RNA in VTM, whereas less than 10 copies/reaction viral RNA were detected in eSwab®. The positive, negative, and overall concordance between the RNA extraction and the direct method was 84%, 100%, and 85%, respectively, in eSwab® samples, whereas the values were 35%, 100%, and 38%, respectively, in VTM samples. When the clinical samples were diluted 1,000 times, GeneSoC® RT-PCR could detect as low as 1.15 copies/reaction RNA using direct method, and the sensitivity was comparable to that of RNA extraction. Conclusion The combination of the direct method and microfluidic rapid PCR machine GeneSoC® has a high sensitivity for detecting SARS-CoV-2 RNA in clinical samples with eSwab® transport medium. [ABSTRACT FROM AUTHOR]

Abstract Background In a previous retrospective observational study, a 3-day regimen of oseltamivir as post-exposure prophylaxis (PEP) for preventing transmission of influenza in wards was shown to be comparable to 7- to 10-day regimens provided index cases were immediately separated from close contacts. In order to confirm the efficacy of a 3-day regimen, we started to conduct a prospective, multi-center, single-arm trial. Methods This study is a prospective, multi-center, single-arm study designed by the Sectional Meeting of Clinical Study, Japan Infection Prevention and Control Conference for National and Public University Hospitals. Index patients with influenza are prescribed a neuraminidase inhibitor and are discharged immediately or transferred to isolation rooms. The close contacts are given oseltamivir as 75 mg capsules once daily for adults or 2 mg/kg (maximum of 75 mg) once daily for children for 3 days as PEP. All close contacts are monitored for development of influenza for 7 days after starting PEP. Discussion A 3-day regimen of oseltamivir as PEP has advantages over 7- to 10-day regimens in terms of costs, medication adherence and adverse effects. Trial registration The Institutional Review Board of Hokkaido University Hospital for Clinical Research, 015-0518, registered on November 11, 2016. UMIN Clinical Trials Registry, UMIN000024458, disclosed on October 31, 2016. https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000027881 . Japan Registry of Clinical Trials, jRCTs011180015, disclosed on March 14, 2019. https://jrct.niph.go.jp/latest-detail/jRCTs011180015

Abstract 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.

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.

Background Rapid antigen tests are widely used to diagnose influenza. However, despite their simplicity and short turnover time, the sensitivity of these tests is relatively low, and molecular tests with greater sensitivity are being sought. In this study, we developed and clinically evaluated a protocol for the rapid multiplex testing of influenza A and B, using a rapid real-time PCR system, GeneSoC®, that is based on microfluidic thermal cycling technology. Methods The specificity of the developed assay was validated using cultured viral strains of influenza A/B, human metapneumovirus, and respiratory syncytial virus. Analytical sensitivity was evaluated using serially diluted RNA synthesized via in vitro transcription and nasopharyngeal swab samples collected from consecutive patients seeking medical attention for a combination of upper respiratory and general symptoms. Crossvalidation of GeneSoC® based on comparisons with conventional real-time RT-PCR and rapid antigen tests was performed by parallel testing of influenza-positive clinical specimens. Results The GeneSoC® assay detected the target sequences of influenza A and B at minimum concentrations of 38 and 65 copies/µL in reaction, respectively. For the analysis of clinical specimens, the positive, negative, and overall agreement between GeneSoC® RT-PCR and a conventional real-time RT-PCR was in all cases 100%, whereas for the comparison between GeneSoC® RT-PCR and the rapid antigen test, the agreements for positive, negative, and overall findings were 100%, 90.9%, and 95.7%, respectively. The mean time for completing GeneSoC® RT-PCR was 16 min 29 s (95% confidence interval, 16 min 18 s to 16 min 39 s). Conclusion The microfluidic real-time PCR system, GeneSoC®, has an analytical performance comparable to that of conventional real-time RT-PCR with rapid turnover time, and represents a promising alternative to rapid antigen tests for diagnosing influenza A and B. [ABSTRACT FROM AUTHOR]

Background: East Asian desert dust storms that occur during mainly spring are called Asian dust storms (ADS). Our objective was to study the association of pollen and ADS with symptoms of adult asthma patients in Japan. Methods: We designed a telephone survey to investigate the upper and lower respiratory, ocular, and skin symptoms of asthma patients during ADS in February, March, and December on 2009. Peak expiratory flow (PRF) was also measured from February to May. Results: We surveyed 106 patients in February, 101 patients in March, and 103 patients in December. In February and March, Japanese cedar and/or cypress pollen was also in the atmosphere during ADS, but no pollen was identified during December survey. Worsening of upper or lower respiratory, ocular, or skin symptoms was noted by 20.8% of patients in February, 33.7% in March, and 16.5% in December. Worsening of symptoms was significantly more common in March than in February or December. Two patients needed emergency treatment for exacerbation during ADS in March, but no patient needed hospitalization in any period. There was no significant difference of the daily morning PEF/personal best PEF ratio between ADS days and control days. However, in patients with worsening of upper and/or lower respiratory tract symptoms, the daily morning PEF/personal best ratio was significantly associated with the atmospheric level of particulate matter, but not with levels of pollen or other air pollutants. Conclusions: Pollen augmented symptoms in adult asthma patients, but ADS on its own also were able to aggravate symptoms and pulmonary function.

Background: Severe wind storms during spring in East Asia, called Asian dust storms (ADS), have been assessed in the past for their effect on health in Asian countries. Our objective was to study the ADS association with asthma symptoms in adult patients in Japan. Methods: We designed a telephone survey to assess ADS influence on upper and lower respiratory, ocular and cutaneous symptoms in 98 patients with adult asthma from April to May 2007. Peak expiratory flow (PEF) was also measured from February to May. Results: Worsening lower respiratory symptoms were noted by 22 of 98 patients during ADS in April, when Japanese cedar pollen levels also increased. During ADS in May, however, Japanese cedar and cypress pollen levels were not elevated, 11 patients had worsening of lower respiratory symptoms. None required emergency treatment for the exacerbation. Lower respiratory symptoms worsening most were cough and sputum; this was more common in patients with allergic rhinitis or atopy than in those without (P < 0.05). Min%Max differed significantly at 88.7 ± 6.6% during dust dispersion period, defined as the ADS day plus the next 6 days, versus 92.0 ± 5.3% during the 7-day period before a dust storm. Conclusions: We found that ADS aggravated lower respiratory symptoms in adult patients with asthma, but this influence was mild.

Bronchial asthma is a chronic inflammatory disorder of the airways, in which inflammation causes bronchial hyper-responsiveness and flow limitation in the presence of various stimuli. Pulmonary function in asthmatic patients frequently deteriorates between midnight and early morning, which has suggested a role for chronotherapy. Although relationships between bronchial asthma and the function of clock genes remain unclear, some medications given for asthma such as glucocorticoids or β2-adrenoceptor agonists may influence clock genes in vivo. In our studies of clock gene mRNA expressions in human bronchial epithelial cells in vitro and peripheral blood cells in vivo, we demonstrated that glucocorticoid or β2-adrenoceptor agonist treatment strongly induced human Per1 mRNA expression both in vitro and in vivo. Human peripheral blood cells provide a useful indication of peripheral clock gene mRNA expression in vivo. Keywords:: β2-adrenoceptor agonist, cAMP, clock gene, glucocorticoid, Per1

Background The epidermal growth factor receptor (EGFR) is a therapeutic target for patients with nonsmall 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 LKB1 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. [ABSTRACT FROM AUTHOR]

Viral infection generally results in the activation of inducible nitric oxide synthase (iNOS or NOS2) in respiratory epithelial cells by inflammatory cytokines. Activated NOS2 catalyzes synthesis of nitric oxide (NO), which in excess can cause cellular injury. On the other hand, lysophosphatidic acid (LPA), a lipid mediator released from epithelial cells, platelets, and fibroblasts in injured tissue, functions in repair of cell injury. However, details of the mechanism for repair by LPA remain unknown. We demonstrated one effect of LPA favoring repair, specifically inhibition by LPA of cytokine-induced NOS2 protein and mRNA expression by human respiratory epithelial cells in vitro. NO production by LPA-treated, cytokine-stimulated cells was also reduced. These decreases were prevented by Rho kinase inhibition with Y-27632. Thus, down-regulation by LPA of cytokine-induced increases in NOS2 activity is likely to involve a Rho-dependent signaling pathway. Harmful biologic effects of NO in viral respiratory infection might be modified by therapeutic manipulations involving LPA or Rho. (Mol Cell Biochem 262: 51–59, 2004) [ABSTRACT FROM AUTHOR]