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Single-tube one-step gel-based RT-RPA/PCR platform for simple, rapid and highly sensitive molecular detection of HIV.

Background: The semi-dry dot-blot (SDB) method, a diagnostic procedure for detecting lymph node (LN) metastases using anti-cytokeratin (CK) antibody, is based on the theory that epithelial components such as CK are not found in normal LNs. Thus, metastases are diagnosed on basis of the presence of CK in lavage fluid of sectioned LNs. We prospectively evaluated novel SDB kits that use a newly developed anti-CK19 antibody and an automatic reader for diagnosing LN metastases in patients with breast cancer. Methods: We obtained 117 LNs dissected from 58 patients with breast cancer between January 2020 and June 2020 at Nagasaki University Hospital. These were sliced at 2-mm intervals and washed with phosphate-buffered saline. Cells suspended in the lavage fluid of sliced LNs were centrifuged and lysed to extract protein. The extracted protein was applied to the SDB kit to diagnose LN metastasis using an automatic reader that evaluates absorbance. Hematoxylin and eosin (H&E) stained washed LNs were blindly examined by pathologists. Diagnoses based on SDB kit and automatic reader findings were compared with diagnoses made by histological examination of paraffin-embedded H&E stained sections of the LNs. Results: Six of the 117 LNs were assessed as positive and 111 as negative by histological examination. With a borderline CK19 absorbance of 50 milli-absorbance (mAbs) for detecting LN metastases excluding isolated tumor cells, the sensitivity, specificity, and overall agreement of the SDB kit were 66.7%, 99.1%, and 97.4%, respectively. Two patients with false-negatives had micrometastases of 0.6 and 0.5 mm in diameter. There was contamination by breast epithelial tissue in one false-positive case. With a borderline CK19 absorbance of 100 mAbs for distinguishing macrometastases from micrometastases, the sensitivity, specificity, and overall agreement of the SDB kit were 100%, 100%, and 100%, respectively. Furthermore, the kits and the automatic reader yielded diagnoses in approximately 20 min at a cost of less than 30 USD. Conclusions: The kits with an automatic reader used in our study were accurate, quick, and cost-effective in diagnosing LN metastases without loss of LN tissue, and were especially useful for detecting distinguish macrometastases. We plan to start a prospective multi-center study to evaluate clinical performance soon. Citation Format: Ryota Otsubo, Hiroshi Yano, Megumi Matsumoto, Aya Tanaka, Ayako Fukushima, Sayaka Kuba, Michi Morita, Naoki Uno, Han-Seung Yoon, Katsunori Yanagihara, Susumu Eguchi, Takeshi Nagayasu. Evaluation of novel diagnostic kits using the semi-dry dot-blot method combined with an automatic reader for detecting metastases in lymph nodes of patients with breast cancer : A single-center prospective study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS1-39.

While clonal heterogeneity has been demonstrated in most cancers, quantitative assessment of individual tumor clones has not been translated to inform clinical practice. A few methods have been developed to investigate the tumor clonality of adult T cell leukemia/lymphoma (ATLL), but currently there is no clinically translatable method available for quantifying individual tumor clones in ATLL patients. Here, we present a methodology to assess the tumor clonality of ATLL and quantify patient-specific tumor clones in a clinical setting. The methodology consists of three steps: (1) selective amplification of restriction fragments containing a human T cell leukemia virus type 1 (HTLV-1) integration site, (2) amplicon deep sequencing to estimate the clonal structure and identify HTLV-1 integration sites of dominant clones, and (3) digital PCR targeting the HTLV-1 integration sites of the dominant clones to quantify specific tumor clones. We successfully tracked individual tumor clones using this approach and demonstrated that each clone had a distinct response to therapies. The procedure is straightforward and clinically feasible, which should facilitate the proper assessment and management of ATLL., Graphical Abstract, A novel procedure is presented for detecting tumor clonality and quantifying individual tumor clones in adult T cell leukemia/lymphoma, which could facilitate the monitoring of patient-specific tumor clones in clinical practice.

Objectives: The accurate detection of severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is essential for the diagnosis of coronavirus disease 2019 (COVID-19). We compared the quantitative RT-PCR results between nasopharyngeal swabs and saliva specimens. Methods: A COVID-19 outbreak occurred on a cruise ship at Nagasaki port, Japan. We obtained 123 nasopharyngeal swabs and saliva each from asymptomatic or mild patients in the late phase of infection. Results: The intervals from the diagnosis to the sampling were 25.5 days for nasopharyngeal swabs and 28.9 days for saliva. The positive rate was 19.5% (24/123) for nasopharyngeal swabs and 38.2% (47/123) for saliva (P = 0.48). The quantified viral copies (mean ± SEM copies/5 μl) were 9.3±2.6 in nasopharyngeal swabs and 920±850 in saliva (P = 0.0006). Conclusions: The advantages of saliva specimens include positive rate improvement and accurate viral load detection. Saliva may be used as a reliable sample for SARS-CoV-2 detection., PLoS ONE, 16(6), art. no. e0252964; 2021

Some macrolides such as 14- and 15-membered macrolides have immunomodulatory effects such as suppression of mucin overproduction. Because a novel macrolide, solithromycin, was developed, we examined whether it suppresses the overexpression of mucin in vitro. A human airway epithelial cell line NCI–H292 was stimulated by Pseudomonas aeruginosa lipopolysaccharides to induce the overproduction of a major mucin, MUC5AC. Treatment with 10 μg/mL of solithromycin significantly inhibited LPS-induced MUC5AC in both mRNA and protein levels as well as a 15-membered macrolide, azithromycin. These findings support that solithromycin has a potential immunomodulatory effect.

The MALDI Biotyper Selective Testing of Antibiotic Resistance–β-Lactamase (MBT STAR-BL) assay enables rapid detection of β-lactamase activity using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. The assay is based on analysis of bacterially induced hydrolysis of β-lactam antibiotics. We investigated the performance of the MBT STAR-BL assay for detecting IMP metallo-β-lactamase (MBL) activity in Enterobacteriaceae. A total of 145 strains (30 Escherichia coli, 43 Klebsiella pneumoniae, and 72 Enterobacter cloacae complex) were evaluated using meropenem hydrolysis assays. The MBT STAR-BL correctly identified all 48 IMP MBL producers as positive, even those exhibiting a low minimal inhibitory concentration (MIC) (1 μg/mL) for meropenem. Conversely, all non–IMP MBL producers, including strains with higher MICs (4 or 8 μg/mL), were correctly identified as negative. The MBT STAR-BL is a rapid, accurate, and reliable system for detecting IMP MBL activity in Enterobacteriaceae.

Objectives To perform surveillance of cfiA-positive Bacteroides fragilis using new subtyping software module, MALDI Biotyper Subtyping Module (MBT Subtyping Module), on MALDI-TOF MS system, and to evaluate the detection ability of the module. Methods cfiA-positive strains were presumed using the module against B. fragilis isolated between 2006 and 2019. The cfiA gene was confirmed using PCR. In cfiA-positive B. fragilis, the insertion sequence (IS) elements were examined and the MBT STAR-BL assay was performed to examine meropenem hydrolysis activity. Results Of the 396 B. fragilis strains included, the MBT Subtyping Module detected 33 presumptive cfiA-positive strains (8.3%), of which 32 harbored the cfiA gene. The sensitivity and specificity of the MBT Subtyping Module for detecting cfiA-positive B. fragilis were 100.0% and 99.7%, respectively. Of the 32 strains harboring the cfiA gene, seven strains possessed IS elements, which were thought to induce high cfiA expression. Meropenem hydrolysis was detected in all seven strains that were positive for both cfiA and IS elements, and they exhibited resistance to meropenem and imipenem. The overall non-susceptibility rates to meropenem and imipenem were 84.8% and 36.4%, respectively, in the 33 presumptive cfiA-positive strains. Conclusion The MBT Subtyping Module can detect cfiA-positive B. fragilis rapidly and accurately, supporting its use for surveillance of cfiA-positive B. fragilis in clinical settings.

This retrospective study evaluated stored nasopharyngeal swab samples from Japanese patients with influenza-like illness during the 2019/2020 season. We aimed to determine whether COVID-19 had spread in the community before the first confirmed case. The period of influenza season during 2019/2020 in Nagasaki was shorter than in previous influenza seasons. When the first COVID-19 case was reported in Nagasaki prefecture, the number of influenza cases were very low. No positive results for SARS-CoV-2 were detected in 182 samples that were obtained from adult outpatients. Our results revealed no large-scale spread of COVID-19 in the community before the first confirmed case., Microbes Infection, 23(4-5), art. no. 104812; 2022

We evaluated a novel transcription-reverse transcription concerted reaction (TRC) assay that can detect influenza A and B within 15 min using nasopharyngeal swab and gargle samples obtained from patients with influenza-like illness, between January and March 2018 and between January and March 2019. Based on the combined RT-PCR and sequencing results, in the nasal swabs, the sensitivity and specificity of TRC for detecting influenza were calculated as 1.000 and 1.000, respectively. In the gargle samples, the sensitivity and specificity of TRC were 0.946 and 1.000, respectively. The TRC assay showed comparable performance to RT-PCR in the detection of influenza viruses., European Journal of Clinical Microbiology & Infectious Diseases, Article in Press.

Influenza is self-limited in otherwise healthy individuals but associated with increased morbidity and mortality in patients at high risk. Diagnosis of influenza infection can be made clinically without laboratory testing in patients who are not at risk and do not require hospital admission during an outbreak that has already been determined to be caused by influenza. However, influenza testing is indicated for patients at high risk for influenza complications, because test results are anticipated to influence clinical management. Influenza testing is also helpful in identifying the cause of outbreaks of respiratory illness. Rapid influenza diagnostic tests (RIDTs), which detect viral antigens in respiratory specimens, are commonly used in clinical practice but problematic because of their limited sensitivity. RIDTs will be replaced by molecular assays to minimize false-negative results.

Surgical antibiotic prophylaxis (SAP) is recommended for the prevention of surgical site infections. However, there is a concern about adverse effects of SAP, such as antibiotic-associated diarrhea (AAD). To prevent AAD, administration of probiotics has been investigated. Although recent advances in next-generation sequencing makes it possible to analyze the gut microbiome, the effect of probiotics on the gut microbiome in the patients with SAP remains unknown. To test a hypothesis that SAP influences the gut microbiome and probiotics prevent the influence, a randomized controlled study was conducted with patients who underwent spinal surgery at Nagasaki University Hospital. After obtaining informed consent, the patients were automatically classified into the non-probiotics group and the probiotics group. In the probiotics group, the patients took 1 g of Enterococcus faecium 129 BIO 3B-R, 3 times a day on postoperative days (PODs) 1–5. The feces of all patients were sampled before administration of SAP and on PODs 5 and 10. We compared alpha and beta diversity and differential abundance analysis of the gut microbiome before and after SAP. During the study period, a total of 33 patients were evaluated, comprising 17 patients in the non-probiotics group and 16 in the probiotics group. There was no significant difference between the groups regarding patient characteristics. In alpha and beta diversity, there were no significant differences among all combinations. In differential abundance analysis at operational taxonomic unit level, Streptococcus gallolyticus and Roseburia were significantly increased in the non-probiotics group and significantly decreased in the probiotics group., Journal of Infection and Chemotherapy, 26(8), pp.795-801; 2020

The MALDI Biotyper Selective Testing of Antibiotic Resistance?β-Lactamase (MBT STAR-BL) assay, which analyzes bacterial induced hydrolysis of cefotaxime using MALDI-TOF MS, correctly identified 100.0% of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae as positive and 94.7% of non-ESBL producers as negative in 80 strains tested., Journal of Microbiological Methods, 160, pp.154-156; 2019

The Verigene®Clostridium difficile nucleic acid test (Verigene® CDF test) is an automatic and rapid detection system for the genes encoding tcdA, tcdB, binary toxin, and the single nucleotide deletion at base pair 117 in the tcdC based on microarray and PCR amplification. We compared the performance of the Verigene® CDF test to that of two enzyme immunoassays, C. DIFF QUIK CHEK COMPLETE and X/Pect Toxin A/B, using 118 specimens. We found overall concordance rates of 81.4% and 78.8% between C. DIFF QUIK CHEK COMPLETE and Verigene® CDF test, and X/Pect Toxin A/B and Verigene® CDF test. The Verigene® CDF test showed the highest sensitivity (93.9%) and had a specificity of 96.5%. The sensitivity and specificity were respectively 45.5 and 94.1% for C. DIFF QUIK CHEK COMPLETE and 27.3 and 100.0% for X/Pect Toxin A/B. These results indicated that the Verigene® CDF test was highly accurate for the detection of C. difficile toxin in fecal specimens and supported its use in daily diagnostic practice.

Genome-wide association studies (GWASs) have reported that human leukocyte antigen (HLA) variants are associated with chronic hepatitis B, spontaneous hepatitis B virus (HBV) clearance, and response to hepatitis B vaccine. Single nucleotide polymorphisms (SNPs) in HLA-DP (rs9277535 and rs3077) and HLA-DQ (rs2856718 and rs7453920) have been repeatedly associated with chronic hepatitis B and spontaneous HBV clearance. However, the data on the SNPs associated with response to hepatitis B vaccine are inconclusive. The objective of this study was to determine whether these four HLA SNPs that have been identified as risk loci for chronic HBV infection are associated with response to hepatitis B vaccine in a Japanese population. We enrolled 278 medical students who received hepatitis B vaccination and measured anti-hepatitis B surface (HBs) antibody titers 1month after a three-dose vaccination series. We found that rs9277535 and rs3077 in HLA-DP were strongly associated with response to hepatitis B vaccine (odds ratio [OR]=0.31 and 0.32, P=0.004 and 0.010, respectively). These two SNPs were significantly associated with anti-HBs titers in an allele-dependent manner. On the other hand, rs2856718 and rs7453920 in HLA-DQ were not associated with response to hepatitis B vaccine. These results indicate that rs9277535 and rs3077 in HLA-DP are the major determinants of response to hepatitis B vaccine, whereas rs2856718 and rs7453920 in HLA-DQ have little effect on the immune response to hepatitis B vaccine.

Purpose. Fluoroquinolone resistance (FQ-r) in extended-spectrum β-lactamase (ESBL) producers is an urgent health concern in countries where ESBL-producing K. pneumoniae (ESBL-Kpn) is prevalent. We investigated FQ-r in Japan where ESBL-Kpn is less prevalent. Methodology. Clinical ESBL-Kpn isolates from 2011 to 2013 were collected in Nagasaki University Hospital. The ESBL genotypes included CTX-M-15, and the mechanisms of FQ-r through plasmid-mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining regions (QRDRs) were examined. Clonality was analysed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and multi-locus sequence typing was performed on selected isolates. Results/Key findings. Thirty ESBL-Kpn isolates, including seven levofloxacin-resistant isolates, were obtained from different patients. An increase in CTX-M-15-producing strains was observed during the study period (0/11 in 2011, 3/8 in 2012, and 5/11 in 2013). PMQR was detected in 53.3?% of the isolates and aac-(6′)-Ib-cr was the most common (36.7?%). ST15 was observed in 60.0?% of the isolates, and for the most predominant ERIC-PCR profiles, 62.5?% of the isolates possessed the CTX-M-15 genotype and 71.4?% were levofloxacin-resistant. Levofloxacin-resistance was significantly more common in CTX-M-15 isolates (62.5?%) compared to non-CTX-M-15 isolates (9.1?%). Three QRDR mutations and aac(6′)-Ib-cr, but not qnrB and qnrS, were significantly enriched in the CTX-M-15 isolates (100.0?%) compared to the non-CTX-M-15 isolates (13.6?%). Conclusion. Cumulatively, these results indicate that the epidemic strain, the CTX-M-15-producing K. pneumoniae ST15, is covertly spreading even when ESBL producers are not prevalent. Monitoring these epidemic strains and ESBLs in general is important for quickly identifying health crises and minimizing future risks from FQ-r ESBL-Kpn., Journal of Medical Microbiology, 66(10), pp.1476-1482; 2017

The innate immune system plays an important role in early immunity against respiratory tract infection. Although airway epithelial cells produce mucus to eliminate pathogens and irritants, hypersecretion of mucus is harmful for the host as it may cause airway obstruction and inhibit influx of antimicrobial agents. It has been reported that several antimicrobial agents have an immunomodulatory effect in vitro and in vivo, but little is known about whether tedizolid, a novel oxazolidinone, can modulate immune responses. In this study, we evaluated whether tedizolid can suppress MUC5AC production in human airway epithelial cells stimulated by methicillin-resistant Staphylococcus aureus (MRSA). Compared with the control, tedizolid significantly inhibited MUC5AC protein production and mRNA overexpression at concentrations of both 2 and 10 μg/mL (representative of trough and peak concentrations in human epithelial lining fluid). Among the mitogen-activated protein kinase inhibitors tested, only extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation was inhibited by tedizolid as indicated by western blot analysis. These results indicate that tedizolid inhibits the overproduction of MUC5AC protein by inhibiting phosphorylation of ERK1/2. This study revealed that tedizolid suppresses excessive mucin production in human airway epithelial cells. The immunomodulatory effect of tedizolid may improve outcomes in patients with severe respiratory infectious diseases caused by MRSA.

Screening of blood for human T-cell lymphotropic virus type 1 and type 2 (HTLV-1 and -2, respectively) is important to diagnose and prevent infection and ensure the safety of blood supplies. The Elecsys HTLV-I/II assay is a newly developed, electrochemiluminescence screening assay for the detection of HTLV-1/2 infection. The sensitivity and specificity of the Elecsys HTLV-I/II assay were determined using well-characterized HTLV-1/2-positive serum and plasma samples and routine diagnostic and blood donor samples expected to be HTLV negative, respectively. These results were compared with those for at least one of the following CE-marked assays at seven independent laboratories and the Roche Diagnostics facility in Penzberg, Germany: Abbott Architect rHTLV-I/II, Ortho Avioq HTLV-I/II Microelisa system, Abbott Prism HTLV-I/HTLV-II, and DiaSorin Murex HTLV I+II. Fujirebio INNO-LIA HTLV-I/II Score was used as a confirmatory assay. The Elecsys HTLV-I/II, Abbott Architect rHTLV-I/II, and Abbott Prism HTLV-I/HTLV-II assays detected all HTLV-1/2-positive samples (sensitivity, 100%). Sensitivity for Ortho Avioq HTLV-I/II was 98.63%. The Elecsys HTLV-I/II assay had a specificity of 99.95% in blood donor samples, which was comparable to results for the other assays (range, 99.91 to 100%). In routine diagnostic samples, the specificity of the Elecsys HTLV-I/II assay was 99.83%, compared with 99.70% for Abbott Architect rHTLV-I/II. Specificity for the Elecsys HTLV-I/II assay in potentially cross-reactive samples was 100%, compared with 99.0% for Ortho Avioq HTLV-I/II and 99.2% for DiaSorin Murex HTLV I+II. The Elecsys HTLV-I/II assay has the sensitivity and specificity to support its use as a routine screening assay for detecting HTLV infection.

The alteration of the microbial community in the upper respiratory tract (URT) can contribute to the colonization and invasion of respiratory pathogens. However, there are no studies regarding whether the characteristics of the URT microbiota can be affected by infections in lower respiratory tract (LRT). To elucidate the microbial profiles of the URT during pneumonia, the oral, nasal, and lung microbiota was evaluated at the early phase in a murine pneumonia model by direct intratracheal inoculation of Klebsiella pneumoniae. The meta 16S rRNA sequencing of bronchoalveolar lavage fluid after K. pneumoniae inoculation presented alterations in the beta diversity of the microbes, but not in the alpha diversity. At this point, a significant increase in microbial alpha diversity was observed in the oral cavity, but not in the nasal cavity. The significant increase was observed in the family Carnobacteriaceae and family Enterococcaceae. These results suggest that characterizing the microbial community of the respiratory tract may not just involve a simple downstream relationship from the URT to the LRT. The health status of the LRT may influence the oral microbiota. Thus, evaluation of the oral microbiota may contribute towards monitoring lung health; the oral microbiota may act as a diagnostic marker of pneumonia., PLoS ONE, 14(9), art.no.e0222589; 2019

The accurate and standardized diagnosis of cytomegalovirus (CMV) infection is important for immunocom- promised patients. We prospectively evaluated the performance of an automated and standardized real-time polymerase chain reaction (PCR) -based DNA quantification for the detection of CMV. The results of PCR- based analysis were also compared with pp65 antigenemia (Ag) assay in the clinical records. The PCR- based analysis of 144 plasma samples from 26 patients with hematologic diseases detected CMV in 69 (48.0%) samples (range,150-1.28 X 10⁴ copies/mL) while Ag detected CMV in 32(22.2%) samples (range, 1-37/50,000 cells). The number of concordant samples between the two tests was 95(66.0%). There were nine patients who had an Ag-positive period sandwiched by Ag-negative periods and, in all these patients, the Ag-positive period was completely covered by PCR-positive period. These results suggest that PCR can detect CMV more sensitively than Ag. The automated and standardized PCR for detection of CMV can support the appropriate management in patients with risks of CMV infection. [Original].

Background Cefiderocol (CFDC) is a novel cephalosporin with siderophore structure, characterized by transportation through siderophore receptor on outer membrane of Gram-negative bacteria and structural stability against beta-lactamase. The antimicrobial activity against multidrug resistant bacteria is demonstrated in vitro and in vivo. In this study, we aimed to elucidate the in vivo efficacy of CFDC using ventilator-associated pneumonia (VAP) mouse model. Methods The minimum inhibitory concentration (MIC) of CFDC and meropenem (MEPM) against the test Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) isolates were measured by broth microdilution assay. Iron depleted medium was used for CFDC. For VAP mouse models, neutropenia was induced by cyclophosphamide intraperitoneal administration, followed by intubation of sterile tube in the trachea and inoculation of bacterial suspension. PK analysis were performed in infected mice, in order to determine treatment regimens to achieve targeted time above MIC (TAM) of free concentrations in plasma. Treatment was initiated 3 hours post infection and continued up to 120 h for survival analysis. To investigate the bactericidal effect, the mice were sacrificed to count bacterial load in the lung at 48 h and 24 h for VAP-Ab and Pa, respectively. Results MICs(mg/L) of CFDC and MEPM against Ab were 0.5 and 128, and Pa were 0.008 and 16, respectively. The treatment regimens to achieve target MIC were shown in Table 1. In order to assess dose dependency of CFDC, required doses to achieve TAM of 70%, 90%, and 100% were calculated. These doses used in the studies were achievable in human for CFDC, but not for MEPM due to high MICs of the test strains. In treatment study for VAP-Ab, bactericidal effect was achieved at TAM > 70% in CFDC groups, as well as TAM 30% in MEPM group. In VAP-Pa, bactericidal effect was observed at TAM > 90% in CFDC groups, as well as TAM 30% in MEPM group. Table 1.Treatment regimen and free TAM against VAP-Ab and Pa Figure 1. Bacterial load in the lungs of VAP-Ab and Pa Conclusion The efficacy of CFDC against VAP-Ab and Pa were demonstrated in this study. Although 90% free TAM was required for bactericidal effect, CFDC was shown to be effective against carbapenem-resistant Gram-negative pathogens at the recommended clinical dosing regimen. Disclosures Katsunori Yanagihara, MD, PhD, Shionogi & Co.,Ltd. (Grant/Research Support)

Introduction Recently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60?80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. Methods We examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN. JAK2 and CALR mutations were analyzed using melting curve analysis and microchip electrophoresis, respectively. Next, we constructed a test for simultaneous screening of the JAK2 and CALR mutations utilizing high resolution melting (HRM). Results Among 99 MPN cases, 60 possessed the JAK2 mutation alone. Of the 39 MPN cases without the JAK2 mutation, 14 were positive for the CALR mutation, all of which were ET. Using our novel screening test for the JAK2 and CALR mutations by HRM, the concordance rate of conventional analysis with HRM was 96% for the JAK2 mutation and 95% for the CALR mutation. Conclusion Our novel simultaneous screening test for the JAK2 and CALR gene mutations with HRM is useful for diagnosis of MPN., Clinica Chimica Acta, 462, pp.166-173; 2016

Tedizolid (TZD) is a second-generation oxazolidinone and demonstrates potent in-vitro activity against multidrug-resistant Gram-positive bacteria. Phase III studies in patients with acute bacterial skin and skin structure infections (ABSSSI) have demonstrated the non-inferiority of TZD to linezolid (LZD). However, there are only a few studies that show the effect of TZD in pulmonary infections. In this study, we investigated the effect of TZD in a murine model of hematogenous pulmonary infection caused by methicillin-resistant Staphylococcus aureus (MRSA). The mice were treated either twice daily with saline (control), 25 mg/kg of vancomycin (low-VAN), 110 mg/kg of vancomycin (high-VAN), 120 mg/kg of LZD or once daily with 20 mg/kg of TZD. As compared to the control, the low- and high-VAN treatment groups, LZD and TZD significantly improved the survival rate, reduced the bacterial count in the lungs. Furthermore, TZD decreased the area of central bacterial colony zone (CBCZ) at 36 h post-inoculation, compared with the control. In addition, we investigated the immunomodulatory effect of TZD by evaluating the plasma concentrations of the inflammatory cytokines. Although there were no significant differences in the bacterial count in the lungs amongst the drugs at 26 h post-inoculation, TZD and LZD significantly improved the plasma concentrations of TNF-alpha, IL-6 and MIP-2, in comparison with the control. In this study, both TZD and LZD demonstrated antimicrobial and immunomodulatory efficacy in a murine model of hematogenous pulmonary infection caused by MRSA., International Journal of Medical Microbiology, 306(6), pp.421-428; 2016

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti-chemokine (C-C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI-8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T-cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI-8000 on ATL-derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI-8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI-8000-induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI-8000-induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI-8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI-8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway., Cancer Science, 107(8), pp.1124-1133; 2016

Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.

In this study, we investigated all Clostridioides difficile strains isolated from stool samples in Nagasaki University Hospital between January 2012 and December 2014. Toxin genes (tcdA, tcdB and cdtA/cdtB) were analyzed for multiplex PCR in a total of 213 strains. In the toxin gene-positive strain, PCR ribotyping was conducted using capillary gel electrophoresis-based PCR and the Webribo database. Patients’ backgrounds were analyzed by departments, disorders, antimicrobials, and clinical dates. The positive rates of tcdA, tcdB, and cdtA/cdtB genes were 62.9%, 63.4%, and 2.8%, respectively. The most frequent PCR ribotype was 047 (14.1%), followed by 014/0 (11.1%) and 002/0 (8.2%). In univariate analysis, the risk factors for the detection of toxin gene-positive strains in patients were older age (p = 0.0036), over ≥ 65 years old (p = 0.0175), the patients hospitalized at Department of Digestive Surgery (P = 0.0059), higher CRP level (P = 0.0395), and lower albumin level (p = 0.0014). In the multivariate analysis, the risk factor for detection of toxin gene-positive strains was the patients hospitalized at Department of Digestive Surgery (OR; 4.62, 95% CI; 1.18–18.0, p = 0.0274). In this study, the percentage of toxin gene-positive and cdtA/cdtB gene-positive strains was almost the same as that reported in previous studies, but the ribotype was different. In addition, we revealed that the risk factor associated with the detection of toxin gene-positive strains was the patients hospitalized at Department of digestive surgery., Journal of Infection and Chemotherapy, 25(4), pp.262-266; 2019

Background Hemolysis during blood drawing is a common cause of laboratory artifacts. Although circulating cell-free tumor DNA and fetal DNA are currently measured in routine practice, the effect of in vitro hemolysis on the measurement of cell-free DNA (cfDNA) has not been investigated. When in vitro hemolysis occurs, cellular DNA could be released from damaged white blood cells and reduce the fraction of circulating tumor DNA and fetal DNA. Methods Blood from healthy individuals was collected and passed through a narrow needle to cause in vitro hemolysis. Plasma was separated before and after mechanical damage, and concentrations of free hemoglobin and cfDNA of 2 reference genes were measured. Results cfDNA of 2 reference genes and free hemoglobin increased after mechanical damage. A clear correlation between cfDNA and free hemoglobin was observed. Conclusion cfDNA concentrations are higher in hemolyzed plasma. Therefore, the fraction of circulating tumor DNA and fetal DNA can be underestimated in plasma hemolyzed by inappropriate blood collection techniques.

Phenotypic detection of extended-spectrum β-lactamase (ESBL) is important for public health and infection control; however, plasmid-mediated AmpC β-lactamases (pAmpCs) can interfere with the ESBL phenotyping. We focused on Enterobacteriaceae strains that were susceptible to cefepime but had a mildly elevated minimum inhibitory concentration (MIC) of ceftazidime and studied the effect of pAmpC on the ESBL phenotyping in this population. Genotyping of ESBL and pAmpC was performed on 528 clinical isolates of Escherichia coli, Klebsiella spp., and Proteus spp. with a ceftazidime MIC of ≥2 μg/mL and cefepime MIC≤8 μg/mL; these isolates were collected at Nagasaki University Hospital from January 2005 to March 2011. In this sample, 145 isolates (27.5%) tested positive for pAmpC (pAmpC group). The concordance rates of phenotypic and genotypic detection of ESBLs were 69.2% in the pAmpC group and 88.8% in the non-pAmpC group (P=0.04). pAmpC was more commonly detected in isolates with non-CTX-M genes (5/53, 9.4%) than in isolates with CTX-M genes (8/121, 6.6%). Our data suggest that the presence of pAmpC increases the false negative detection of ESBL. When ESBL phenotyping is used, the underestimation of the prevalence of ESBL producers should be taken into account.

This study investigated the molecular and phenotypic characteristics of carbapenemase-producing Klebsiella pneumoniae, and identified the risk factors underlying its acquisition. We evaluated K. pneumoniae isolated in Nagasaki University Hospital between January 2009 and June 2015. The presence of carbapenemase genes and plasmid characteristics were investigated. We performed multilocus sequence typing (MLST), and generated a dendrogram based on the results of pulsed-field gel electrophoresis (PFGE) for carbapenemase-producing strains. We also performed a case-control study of patients. Of the 88 K. pneumoniae strains that showed minimum inhibitory concentration ≥1 μg/mL for imipenem and/or meropenem, and that were available from our bacterial collection, 18 had the IMP-type carbapenemase gene, all of which were IMP-1 according to sequencing analysis. Strains included seven different sequence types (STs), of which the most common was ST1471. A dendrogram showed the significant similarity of some strains with relationships in PFGE patterns, STs, and the wards in which they were isolated. Plasmid incompatibility group was similar among the IMP-1 producers. Regarding risk factors, multivariate analysis showed that liver disease and previous uses of carbapenems and anti-fungal drugs were significant factors for the acquisition of IMP-1-producing strains. Our results demonstrate that IMP-1 is a major carbapenemase produced by K. pneumoniae. The PFGE results indicated the possibility of transmission in the hospital. The identified risk factors should be considered for appropriate antibiotic therapy and infection-control measures.

Lascufloxacin showed potent activity against Streptococcus pneumoniae with a GyrA or ParC mutation (first-step mutant). The frequency of selecting resistant strains tended to be lower for lascufloxacin than for levofloxacin and garenoxacin after drug exposure in first-step mutants but was similar in the comparison between lascufloxacin and moxifloxacin. The increase in MIC was smaller for lascufloxacin than for levofloxacin, garenoxacin, and moxifloxacin when clinical strains with only ParC mutations were exposed to the corresponding drug., Antimicrobial Agents and Chemotherapy, 62(4), 01971-17; 2018

Adult T-cell leukemia-lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target. This report first describes the effectiveness of a novel HSP90 inhibitor NVP-AUY922 to adult T-cell leukemia-lymphoma (ATL) cells., Cancer Science, 105(12), pp.1601-1608; 2014

Meropenem-susceptible and -resistant Aeromonas dhakensis isolates from blood cultures of a fatal case of septicemia were analyzed. The two isolates were homologous and gene expression of metallo-β-lactamase in the resistant strain was upregulated. Physicians should be aware of the possibility of the induction of carbapenem-resistance, following the use of carbapenems in the treatment of Aeromonas infection.

BD Phoenix? is an automated bacterial identification and susceptibility testing system. Here, its performance in screening IMP-producing Enterobacteriaceae was evaluated. The system identified 97.8% of IMP producers as being nonsusceptible to imipenem or meropenem, which was higher than that identified by the broth microdilution method (91.3%, imipenem; 41.3%, meropenem)., Journal of Microbiological Methods, 145, pp.47-49; 2018

Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for detecting genomic structural variants. The technique is based on hybridization and ligation, followed by amplification of the ligation products. Therefore, ligation is considered a fundamental process that determines the feasibility and fidelity of MLPA. However, despite the widespread use of this technique, its reaction mechanism has not been fully analyzed. Herein, we describe a ligation-independent pathway for MLPA and introduce a ligation-independent probe amplification system that can be used to obtain amplified products without the hybridization and ligation processes. Fragment analysis revealed that the ligation-independent pathway is functional and that the capacity to discriminate single nucleotides with MLPA does not depend on ligation. These findings indicate that the feasibility and fidelity of MLPA do not rely on ligation., Analytical Sciences, 30(8), pp.805-810; 2014

Background: Carbapenems, fluoroquinolones (FQs), and aminoglycosides (AGs) are key drugs for treating Pseudomonas aeruginosa infections, and accumulation of drug resistances make antibiotic therapy difficult. Methods: We evaluated 169 patients with imipenem (IPM)-resistant P. aeruginosa and compared patient background and microbiological characteristics between groups with or without FQ resistance. Similar analyses were performed for AG. Results: Of the 169 IPM-resistant strains, 39.1% showed resistance to FQs and 7.1% to AGs. The frequency of exposure to FQs within 90 days previously was higher in the group with FQ resistance (45.5%) than in the group without FQ resistance (13.6%). Similarly, 33.3% of patients in the group with AG resistance had been previously administered AGs, higher than the 7.6% of patients without AG resistance. Frequencies of metallo-β-lactamase (MBL) production were higher in the group with FQ or AG resistance (16.7% or 33.3%) than in the group without FQ or AG resistance (2.9% or 6.4%). Multivariate analyses showed exposures to FQs or AGs were related to the respective resistances. MBL production was a common factor for resistance to FQs or AGs, in addition to IPM-resistant P. aeruginosa. Conclusion: As well as promoting appropriate use of antibiotics, MBL production should be detected as a target of intervention for infection control., The Open Microbiology Journal, 11, pp.92-97; 2017

The Verigene

Background Arbekacin is an aminoglycoside that shows strong antimicrobial activity against Gram-positive bacteria, including MRSA, as well as Pseudomonas aeruginosa . The therapeutic effectiveness of arbekacin is directly related to C max at the infection site. To maximize drug delivery to the respiratory tract and minimize the systemic toxicity, arbekacin optimized for inhalation, ME1100, is under development. In this study, we investigated the efficacy and pharmacokinetics of ME1100 in a murine model of ventilator-associated pneumonia caused by P. aeruginosa by using a customized investigational nebulizer system. Methods The mice were treated for 5 min, once daily, with placebo, 3, 10 or 30 mg/mL ME1100 or 30 mg/mL amikacin. Results In the survival study, the survival rate was significantly improved in the 10 and 30 mg/mL ME1100 treatment groups compared with that in the placebo group. The number of bacteria in the lungs was significantly lower in the 30 mg/mL ME1100 treatment group at 6 h after the initial treatment, compared with all other groups. In the pharmacokinetic study, the C max in the 30 mg/mL ME1100 treatment group in the epithelial lining fluid (ELF) and plasma was 31.1 and 1.2 mg/L, respectively. Furthermore, we compared the efficacy of ME1100 with that of amikacin. Although there were no significant differences in ELF and plasma concentrations between 30 mg/mL of ME1100 and 30 mg/mL of amikacin, ME1100 significantly improved the survival rate compared with amikacin. Conclusions The results of our study demonstrated the in vivo effectiveness of ME1100 and its superiority to amikacin.

Background TNF-α plays an important role in the pathogenesis of Legionella pneumophila (Lp)-induced pneumonia. Patients undergoing anti-TNF-α therapy are at an increased risk of Lp infection. Lp infects both phagocytic and non-phagocytic cells such as airway epithelial cells; however, the role of TNF-α in airway epithelial cells is unknown. Methods Human airway epithelial cell line NCI-H292 was infected with Lp NUL1 strain. After infection, both intracellular growth of Lp and cell death were evaluated after treating the cells with or without TNF-α. Apoptosis was examined by performing activated caspase-3/7 staining and by using a pan-caspase inhibitor. Results Lp infected and replicated in NCI-H292 cells in a time-dependent manner, and TNF-α treatment of Lp-infected NCI-H292 cells inhibited Lp replication. Inhibitory effects of TNF-α on Lp replication were suppressed after treatment with a TNF-α-neutralizing antibody. Lp infection increased extracellular lactate dehydrogenase levels and decreased the number of living cells. Increased number of Lp-infected NCI-H292 cells showed caspase-3/7 activation, indicating they underwent apoptosis. TNF-α treatment inhibited Lp replication by increasing the apoptosis of NCI-H292 cells. Conclusions Thus, our results suggested that airway epithelial cells were involved in the pathogenesis of Lp infection and that TNF-α played a protective role by inhibiting the intracellular replication of Lp and by increasing the apoptosis of Lp-infected airway epithelial cells. However, Lp infection should be investigated further in patients undergoing anti-TNF-α therapy who develop pneumonia.

We previously uncovered a ligation-independent pathway of multiplex ligation-dependent probe amplification (MLPA) through which products of MLPA could be amplified without both hybridization and ligation reactions. Here, we utilized this pathway to detect an antibiotic resistance mutation of quinolones in Streptococcus pneumoniae.

Meropenem-susceptible and -resistant Aeromonas dhakensis isolates from blood cultures of a fatal case of septicemia were analyzed. The two isolates were homologous and gene expression of metallo-β-lactamase in the resistant strain was upregulated. Physicians should be aware of the possibility of the induction of carbapenem-resistance, following the use of carbapenems in the treatment of Aeromonas infection.

To study the influence of retinal adaptation on oscillatory potential (OP) using repeated-flash electroretinography.Subjects were 28 adult eyes with normal retinas. Four stimuli (four flashes) of white light from a light-emitting diode built into a contact lens that also served as the recording electrode were presented at 5-s intervals after 30 min of dark adaptation (DA) and then after 10 min of light adaptation (LA). Recordings were made without background light.Four OPs (O1, O2, O3, and O4) were recordable. After DA, amplitudes of O1 and O4 decreased with subsequent flashes, whereas those of O2 increased after the second flash. After LA, amplitudes of O3 and O4 were smaller than after DA.Amplitude and implicit time of OPs were influenced by retinal adaptation. Among all OPs, O2 showed unique characteristics in course of retinal adaptation.

Mesoporous anodic oxidized alumina (MAOA) capillary tubes with and without a barrier layer have been synthesized by applying a pulse-sequential voltage. The single gas permeances at an elevated temperature and the thermal and hydrothermal stabilities of MAOA were investigated. A highly oriented radial mesopore channel with pore sizes from 40 to 4 nm was formed in the MAOA tubes. Micropores with sizes from 0.4 to 0.8 nm were formed in the barrier layer. The H2 permeance of MAOA with a barrier layer (barrier type) was approximately 540 times lower than that of MAOA without a barrier layer (block type) at 773 K. The H2/N2 permselectivity of the barrier type in the temperature range from 333 to 673 K was 3.4; those of the barrier type at 773 and 823 K were 4.4 and 11, respectively. On the other hand, the H2/N2 permselectivities of the block type were from 3.1 to 3.6 in the temperature range from 333 to 773 K. The H2 permeance and the H2/N2 permselectivity of the amorphous silica membrane on the block type wer...

Methicillin-resistant Staphylococcus aureus (MRSA) is prevalent in Japan, and the Staphylococcus cassette chromosome mec (SCCmec) type II is common among hospital-acquired MRSA isolates. Information pertaining to MRSA characteristics is limited, including SCCmec types, in primary or secondary care facilities. A total of 128 MRSA isolates (90 skin and soft tissue isolates and 38 blood isolates) were collected at a secondary care facility, Kawatana Medical Center, from 2005 to 2011. Antimicrobial susceptibility testing for anti-MRSA antibiotics and molecular testing for SCCmec and virulence genes (tst, sec, etb, lukS/F-PV) were performed. Strains positive for lukS/F-PV were analyzed by multilocus sequence typing and phage open-reading frame typing. SCCmec typing in skin and soft tissue isolates revealed that 65.6% had type IV, 22.2% had type II, 8.9% had type I, and 3.3% had type III. In blood isolates, 50.0% had type IV, 47.4% had type II, and 2.6% had type III. Minimum inhibitory concentrations, MIC(50)/MIC(90), against vancomycin, teicoplanin, linezolid, and arbekacin increased slightly in SCCmec II isolates from skin and soft tissue. MICs against daptomycin were similar between sites of isolation. SCCmec type II isolates possess tst and sec genes at a greater frequently than SCCmec type IV isolates. Four lukS/F-PV-positive isolates were divided into two clonal patterns and USA300 was not included. In conclusion, SCCmec type IV was dominant in blood, skin, and soft tissue isolates in a secondary care facility in Japan. Because antimicrobial susceptibility varies with the SCCmec type, SCCmec typing of clinical isolates should be monitored in primary or secondary care facilities.

The Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN) is a microarray-based assay that enables rapid detection of 9 common Gram-negative bacteria and 6 resistance determinants directly from positive blood cultures. We compared the performance of BC-GN with currently used automated systems, testing 141 clinical blood cultures and 205 spiked blood cultures. For identification of BC-GN target organisms in clinical and spiked blood cultures, the BC-GN assay showed 98.5% (130/132) and 98.9% (182/184) concordance, respectively. Of 140 resistance genes positively detected in clinical and spiked blood cultures with the BC-GN test, 139 (99.3%) were confirmed by PCR, and the detection results were consistent with the resistance phenotypes observed. The BC-GN assay, thus, can potentially improve care for sepsis patients by enabling timely detection and targeted antimicrobial therapy.