Clinical Features of Human Metapneumovirus Pneumonia in Non-Immunocompromised Patients: An Investigation of Three Long-Term Care Facility Outbreaks.

Background: Several studies have reported outbreaks due to human metapneumovirus (hMPV) in long-term care facilities (LTCF) for the elderly. However, most of these reports are epidemiological studies and do not investigate the clinical features of hMPV pneumonia. Methods: Three independent outbreaks of hMPV occurred at separate LTCF for intellectually challenged and elderly residents. A retrospective evaluation of hMPV pneumonia and its clinical and radiological features was conducted using available medical records and data. Results: In 105 hMPV infections, 49% of patients developed pneumonia. The median age of pneumonia cases was significantly higher than non-pneumonia cases (P < .001). Clinical manifestations of hMPV pneumonia included high fever, wheezing in 43%, and respiratory failure in 31% of patients. An elevated number of white blood cells as well as increased levels of C-reactive protein, creatine phosphokinase, and both aspartate and alanine transaminases was also observed among pneumonia cases. Evaluation of chest imaging revealed proximal bronchial wall thickenings radiating outward from the hilum in most patients. Conclusions: The aforementioned characteristics should be considered as representative of hMPV pneumonia. Patients presenting with these features should have laboratory testing performed for prompt diagnosis.

Phosphorylation of epidermal growth factor receptor at serine 1047 in cultured lung alveolar epithelial cells by bradykinin B2 receptor stimulation.

Accumulating evidence indicates that epidermal growth factor receptor (EGFR) is desensitized by phosphorylation of serine 1047 (Ser1047). We and other groups have reported that stimulation of a receptor of tumor-necrosis factor α (TNFα) and Toll-like receptor 5 (TLR5) induced the phosphorylation of Ser1047 through activation of p38 mitogen-activated protein kinase (p38 MAPK) in cultured lung alveolar epithelial A549 cells. However, phosphorylation of EGFR at Ser1047 by stimulation of any G-protein coupled receptors (GPCRs) has not been reported in any cultured cells. In the present study, we first confirmed that A549 cells expressed bradykinin (BK) B2 receptor, and then, we examined whether BK treatment of A549 cells activated MAPKs and induced the phosphorylation of EGFR at Ser1047. Immunoblotting analysis and reporter gene assays indicated that BK activated the pathways of extracellular signal-regulated kinase (ERK) and p38 MAPK. Inhibitor studies suggested that Gq/11 was mainly involved in the activation of ERK and p38 MAPK. We found that stimulation of the BK B2 receptor, but not the BK B1 receptor, induced phosphorylation of EGFR at Ser1047. Pharmacological experiments indicated that both ERK and p38 MAPK were involved in the phosphorylation of EGFR. These results strongly suggested that BK regulates EGFR functions in lung alveolar epithelial cells. In addition, we found that BK treatment increased the mRNA level of dual specificity MAPK phosphatase 5 (DUSP5) in an ERK-dependent manner, which suggested that a negative feedback mechanism of ERK existed in the cells.

The clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato-oncology unit.

Although many reports have already shown RSV outbreaks among hemato-oncology patients, genomic studies detecting similar RSV strains prior to an outbreak in the hospital are rare. In 2014, the University of the Ryukyus hospital hemato-oncology unit experienced, and successfully managed, a respiratory syncytial virus (RSV) nosocomial outbreak. During the outbreak investigation, genotyping and phylogenetic analysis was used to identify a potential source for the outbreak. Nasopharyngeal swabs were tested for RSV using three tests: (1) rapid antigen test (RAT); (2) reverse transcriptase polymerase chain reaction (PCR); or (3) quantitative PCR (RT-qPCR); a positive PCR reaction was considered a confirmed case of RSV. Phylogenetic analysis of the G protein was performed for outbreak and reference samples from non-outbreak periods of the same year. In total, 12 confirmed cases were identified, including 8 hemato-oncology patients. Patient samples were collected weekly, until all confirmed RSV cases returned RSV negative test results. Median time of suspected viral shedding was 16 days (n = 5, range: 8-37 days). Sensitivity and specificity of the RAT compared with RT-qPCR were 30% and 91% (n = 42). Phylogenetic analysis revealed nine genetically identical strains; eight occurring during the outbreak time period and one strain was detected 1 month prior. A genetically similar RSV detected 1 month before is considered one potential source of this outbreak. As such, healthcare providers should always enforce standard precautions, especially in the hemato-oncology unit.

Evaluation of Anyplex™ II RV16 and RB5 real-time RT-PCR compared to Seeplex® RV15 OneStep ACE and PneumoBacter ACE for the simultaneous detection of upper respiratory pathogens.

This prospective study was performed to evaluate and compare the performance of the multiplex PCR Seeplex® assays and Anyplex™ II assays. From May 2014 until April 2016, a total of 247 respiratory samples were collected in Okinawa, Japan. Multiple respiratory pathogens were detected in 37% of patients with positive results. The most prevalent pathogens were influenza A virus and respiratory syncytial virus B. Despite minor differences in capabilities, both the Seeplex® assays and Anyplex™ II assays can be easily implemented in diagnostic or research laboratories to optimize the detection and management of respiratory pathogen induced diseases.

Increased expression of EGR1 and KLF4 by polysulfide via activation of the ERK1/2 and ERK5 pathways in cultured intestinal epithelial cells.

Sodium trisulfide (Na2S3) releases hydrogen polysulfide (H2Sn) and is useful for the investigation of the effects of H2Sn on the cell functions. In the present study, we first examined the effects of Na2S3 on the gene expression of IEC-6 cells, a rat intestinal epithelial cell line. Microarray analysis and reverse transcription-polymerase chain reaction analysis revealed that Na2S3 increased the gene expression of early growth response 1 (EGR1) and Kruppel-like transcription factor 4 (KLF4). It was interesting that U0126, an inhibitor of the activation of extracellular signal-regulated kinase 1 (ERK1), ERK2, and ERK5, inhibited the Na2S3-induced gene expression of EGR1 and KLF4. Na2S3 activated ERK1 and ERK2 (ERK1/2) within 15 min. In addition to ERK1/2, Na2S3 activated ERK5. We noticed that the electrophoretic mobility of ERK5 was decreased after Na2S3 treatment. Phos-tag analysis and in vitro dephosphorylation of the cell extracts indicated that the gel-shift of ERK5 was due to its phosphorylation. The gel-shift of ERK5 was inhibited completely by both U0126 and ERK5-IN-1, a specific inhibitor of ERK5. From these results, we concluded that the gel-shift of ERK5 was induced through autophosphorylation by activated ERK5 after Na2S3 treatment. The present study suggested that H2Sn affected various functions of intestinal epithelial cells through the activation of the ERK1/2 and ERK5 pathways.

Regulation of epidermal growth factor receptor expression and morphology of lung epithelial cells by interleukin-1ß.

Accumulating evidences suggested that the overactivation of epidermal growth factor receptor (EGFR) was involved in the development of adult respiratory distress syndrome and pulmonary fibrosis. Elucidation of the mechanisms that regulate EGFR residence on the plasma membrane during inflammatory lung conditions is important for identifying potential therapies. We have demonstrated that flagellin phosphorylated EGFR at Ser1047 and induced transient EGFR internalization. In this study, we examined the molecular pathway and effect of interleukin 1 beta (IL-1ß) on EGFR in alveolar epithelial cells. Treatment of A549 cells with IL-1ß induced the activation of p38 mitogen-activated protein kinase (MAP kinase) and MAP kinase-activated protein kinase-2 (MAPKAPK-2), as well as EGFR phosphorylation at serine 1047. Both MAPKAPK-2 activation and EGFR phosphorylation were inhibited by SB203580, a p38 MAP kinase inhibitor. In addition, MK2a inhibitor (a MAPKAPK-2 inhibitor) suppressed EGFR phosphorylation. Assessment of the biotinylation of cell surface proteins indicated that IL-1ß induced EGFR internalization. Furthermore, long-term treatment of A549 cells with IL-1ß caused morphological changes and loss of cell-cell contact. Moreover, IL-1ß augmented the effect of transforming growth factor beta 1 on the epithelial-mesenchymal transition. These results suggested that IL-1ß regulates EGFR functions and induces morphological changes of alveolar epithelial cells.

Evaluation of a multiplex PCR assay for detection of cytomegalovirus in stool samples from patients with ulcerative colitis.

AIM: To evaluate a multiplex PCR assay for the detection of bacterial and viral enteropathogens in stool samples from patients with ulcerative colitis (UC). METHODS: We prospectively analyzed 300 individuals, including immunocompetent patients, immunocompromised patients, and patients with UC. Stool samples were collected from the recto-sigmoid region of the colon by endoscopy. The samples were qualitatively analyzed for bacterial and viral enteropathogens with a multiplex PCR assay using a Seeplex(®) Kit. Additional clinical and laboratory data were collected from the medical records. RESULTS: A multiplex PCR assay detected 397 pathogens (191 bacteria and 206 viruses) in 215 samples (71.7%). The most frequently detected bacteria were Escherichia coli H7, 85 (28.3%); followed by Aeromonas spp., 43 (14.3%); and Clostridium perfringens, 36 (12.0%) samples. The most prevalent viruses were Epstein-Barr virus (EBV), 90 (30.0%); followed by human herpes virus-6 (HHV-6), 53 (17.7%); and cytomegalovirus (CMV), 37 (12.3%) samples. The prevalence rate of CMV infection was significantly higher in the immunocompromised group than in the immunocompetent group (P < 0.01). CMV infection was more common in patients with UC (26/71; 36.6%) than in the immunocompetent patients excluding UC (6/188; 3.2%) (P < 0.01). CMV infection was more prevalent in UC active patients (25/58; 43.1%) than in UC inactive patients (1/13; 7.7%) (P < 0.05). Among 4 groups which defined by the UC activity and immunosuppressive drugs, the prevalence rate of CMV infection was highest in the UC active patients with immunosuppressive drugs (19/34; 55.8%). Epstein-Barr virus (EBV) infection was more common in the immunocompromised patients excluding UC (18/41; 43.9%) than in the immunocompetent patients excluding UC (47/188; 25.0%) (P < 0.05). The simultaneous presence of CMV and EBV and/or HHV6 in UC active patients (14/58; 24.1%) was greater than in immunocompromised patients excluding UC (5/41; 12.2%) (P < 0.05). CONCLUSION: The multiplex PCR assay that was used to analyze the stool samples in this study may serve as a non-invasive approach that can be used to exclude the possibility of CMV infection in patients with active UC who are treated with immunosuppressive therapy.