Difference in TMPRSS2 usage by Delta and Omicron variants of SARS-CoV-2: Implication for a sudden increase among children.

It has been postulated from a combination of evidence that a sudden increase in COVID-19 cases among pediatric patients after onset of the Omicron wave was attributed to a reduced requirement for TMPRSS2-mediated entry in pediatric airways with lower expression levels of TMPRSS2. Epidemic strains were isolated from the indigenous population in an area, and the levels of TMPRSS2 required for Delta and Omicron variants were assessed. As a result, Delta variants proliferated fully in cultures of TMPRSS2-positive Vero cells but not in TMPRSS2-negative Vero cell culture (350-fold, Delta vs 9.6-fold, Omicron). There was no obvious age-dependent selection of Omicron strains affected by the TMPRSS2 (9.6-fold, Adults vs. 12-fold, Children). A phylogenetic tree was generated and Blast searches (up to 100 references) for the spread of strains in the study area showed that each strain had almost identical homology (>99.5%) with foreign isolates, although indigenous strains had obvious differences from each other. This suggested that the differences had been present abroad for a long period. Therefore, the lower requirement for TMPRSS2 by Omicron strains might be applicable to epidemic strains globally. In conclusion, the property of TMPRSS2-independent cleavage makes Omicron proliferate with ease and allows epidemics among children with fewer TMPRSS2 on epithelial surfaces of the respiratory organs.

[Fatal invasive pneumococcal disease developed 14 years after allogeneic hematopoietic stem cell transplantation in a patient with myelodysplastic syndrome].

Invasive pneumococcal diseases (IPDs) after allogeneic hematopoietic stem cell transplantation have high fatality rates and often develop late after transplantation. The patient was a 58-year-old female. Fourteen years ago, she underwent bone marrow transplantation from a HLA-DR 1-antigen mismatched unrelated donor for myelodysplastic syndrome. She developed pneumonia, chronic graft-versus-host disease, and hypogammaglobulinemia. She received 23-valent pneumococcal capsular polysaccharide vaccine 11 and 6 years earlier. She was presented to our emergency room with fever. Her blood culture was positive for pneumococcus, and she was diagnosed with an IPD. The patient received antibiotic treatment but died on the third day of hospitalization. Because of its seriousness, pneumococcal infection should receive attention even 10 or more years after transplantation. Preventive approaches such as vaccination and early intervention at the time of diagnosis are important.

Rapid Multiplex RT-PCR for Influenza A and B by Genesoc®, a Microfluidic PCR System.

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. Cross-validation 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.

Clinical characteristics and treatment outcomes of carbapenem-resistant Enterobacterales infections in Japan.

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.

Three-day regimen of oseltamivir for post-exposure prophylaxis of influenza in hospital wards: a study protocol for a prospective, multi-center, single-arm trial.

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.

Evaluation of infections in orthopedic patients using next-generation sequencing.

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.

Peritoneal dialysis-associated peritonitis caused by Mycobacteroides massiliense: the first case and review of the literature.

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.

Effect of Cetuximab and EGFR Small Interfering RNA Combination Treatment in NSCLC Cell Lines with Wild Type EGFR and Use of KRAS as a Possible Biomarker for Treatment Responsiveness.

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.

Evaluation of antigen-positive toxin-negative enzyme immunoassay results for the diagnosis of toxigenic Clostridium difficile infection.

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.

Splenic Infarction in Acute Cytomegalovirus and Human Parvovirus Concomitant Infection.

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.

γ-Tocotrienol Inhibits TGF-ß1-Induced Contractile Phenotype Expression of Human Airway Smooth Muscle Cells.

BACKGROUND: Tocotrienols, members of the vitamin E family, exist in four different isoforms (α, ß, γ and δ tocotrienol) that have can be protective against brain damage, as well as having anticancer effects in vivo and in vitro. We have shown that γ-tocotrienol inhibits human airway smooth muscle cell proliferation and migration induced by platelet-derived growth factor (PDGF)-BB by suppressing RhoA activation. In this study, we tested whether γ-tocotrienol modulates transforming growth factor (TGF) -ß-induced induction of human airway smooth muscle (ASM) into a contractile phenotype and concomitant synthesis of extracellular matrix proteins. METHODS: ASM cells were stimulated with TGF-ß1 (2 ng/mL) for 48 hours and the effect of γ-tocotrienol (50 µM) on α-smooth muscle actin, fibronectin and collagen I expression was assessed using Western blotting. The signaling pathways involved in TGF-ß1 stimulation were also investigated. RESULTS: TGF-ß1 increased α-smooth muscle actin, fibronectin and collagen Ⅰ abundance by 3- to 5-fold. This response was inhibited significantly by γ-tocotrienol. Furthermore, γ-tocotrienol suppressed RhoA activation, but did not affect Smad2 or Smad3 phosphorylation. CONCLUSION: These results indicate that γ-tocotrienol has potential for benefit in modulating on airway remodeling in asthma, likely via a mechanism involving the suppression of TGF-ß activation of RhoA.

Shewanella algae Bacteremia in an End-stage Renal Disease Patient: A Case Report and Review of the Literature.

A 71-year-old man was admitted because of nausea and abdominal pain. He was receiving an erythropoiesis-stimulating agent for anemia and dysregulated iron metabolism due to stage G5 chronic kidney disease. He had a history of raw fish intake and was diagnosed with infectious enterocolitis, which worsened and led to septic shock. Shewanella putrefaciens grew in the blood culture, but Shewanella algae was identified in a 16S rRNA gene sequence analysis. We herein report a case of S. algae bacteremia believed to have been transmitted orally. We also reviewed previous case reports on Shewanella infection in end-stage renal disease patients.

Properties of Achromobacter xylosoxidans highly resistant to aminoglycoside antibiotics.

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.

Effect of Clarithromycin on the Expression of UL16-Binding Protein 2 in Human Cells.

BACKGROUND: Clarithromycin is a macrolide antibiotic that possesses anti-inflammatory and immunomodulatory properties. Although recent data suggests that macrolide antibiotics enhance Pseudomonas aeruginosa clearance from the lung, involving natural killer (NK) T cells in this process by activating the NKG2D-NKG2D ligand system, the precise underlying mechanism is still unclear. In this study, we examined the effect of clarithromycin on a potent NKG2D ligand, UL16-binding protein 2 (ULBP2), in the lung and its shedding mechanism. METHODS: The gene expressions of ULBP2 and the shredder proteinases of ULBP2, a disintegrin and metalloproteinase domain 10 (ADAM10) and ADAM17, were measured using real-time PCR. The cell surface ULBP2 expression was measured by flow cytometry. The amount of solubilized ULBP2 (sULBP2) was measured using an ELISA. The activity of ADAM17 was examined by measurement of fluorescence intensity from the fluorescence resonance energy transfer peptide substrate cleaved by ADAM17. RESULTS: Clarithromycin significantly induced transcription of ULBP2 and ADAM17 in both A549 and LCSC #2 cells, which endogenously express minimal and abundant levels of ULBP2, respectively. However, there was no significant change on transcription of ADAM10. The same tendency was observed when LCSC #2 cells were treated with tumor necrosis factoralpha processing inhibitor-2 to inhibit ADAM17 activity. The amount of sULBP2 was significantly decreased in both A549 and LCSC #2 cells by treatment with clarithromycin. Finally, clarithromycin significantly inhibited the activity of ADAM17 in LCSC #2 cells. CONCLUSION: These findings suggest that clarithromycin induces ULBP2 expression and reduces the amount of sULBP2, by possibly inhibiting the activity of the potent ULBP2-shedding enzyme ADAM17. Because these changes in ULBP2 and sULBP2 levels could activate NKT cells, this finding might indicate a novel mechanism by which clarithromycin improves the clearance of P. aeruginosa in chronic respiratory diseases.

γ-Tocotrienol reduces human airway smooth muscle cell proliferation and migration.

AIMS: Vitamin E is an antioxidant that occurs in 8 different forms (α, ß, 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.

Respiratory viruses and torque teno virus in adults with acute respiratory infections.

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.

A novel point-of-care system for high-speed real-time polymerase chain reaction testing for epidermal growth factor receptor mutations in bronchial lavage fluids after transbronchial biopsy in patients with non-small cell lung cancer.

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.

A new rapid method for detecting epidermal growth factor receptor mutations in non-small cell lung cancer.

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 <10 min, and all steps could be accomplished in <50 min after sample collection. Thus, our novel PCR method offers a rapid, simple, and less expensive test for EGFR mutations and can be applied as a point-of-care diagnostic test.

Synergistic cell growth inhibition by the combination of amrubicin and Akt-suppressing agents in K-ras mutation-harboring lung adenocarcinoma cells: implication of EGFR tyrosine kinase inhibitors.

Previously we showed that Akt-suppressing agents, combined with amrubicin, synergistically inhibited the growth of small cell lung cancer cells. The combined effects of chemotherapeutic agents and Akt-suppressing agents, including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, were evaluated in A549 lung adenocarcinoma cells harboring K-ras mutation and wild-type EGFR. Only amrubicin and not other chemotherapeutics (cisplatin, pemetrexed and paclitaxel) synergistically inhibited cell growth when combined with an Akt inhibitor, LY294002. The combination of amrubicin and LY294002 enhanced Annexin V binding to cells. A non-specific tyrosine kinase inhibitor, genistein, suppressed Akt and showed synergistic interaction in combination with amrubicin. Two EGFR tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, suppressed Akt activity at clinically achievable concentrations and demonstrated synergism when combined with amrubicin. The suppression of K-ras expression by siRNA interfered with this synergism and inhibited both EGFR and Akt activity in A549 cells. In Ma10 cells, which harbor wild-type EGFR and K-ras, EGFR-TKIs neither suppressed Akt activity nor exhibited such synergism when combined with amrubicin. We concluded that the synergism by the combination of EGFR-TKI and amrubicin is attributable, at least partially, to K-ras mutation in A549 cells. The combination of EGFR-TKI and amrubicin may be a promising treatment for lung cancer with wild-type EGFR and K-ras mutation.

[A case of lung abscess due to Aspergillus viridinutans in a patient with aplastic anemia].

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 ß-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.