Norihisa Yamamoto

Presence of Neutrophil Extracellular Traps and Citrullinated Histone H3 in the Bloodstream of Critically Ill Patients

Neutrophil extracellular traps (NETs), a newly identified immune mechanism, are induced by inflammatory stimuli. Modification by citrullination of histone H3 is thought to be involved in the in vitro formation of NETs. The purposes of this study were to evaluate whether NETs and citrullinated histone H3 (Cit-H3) are present in the bloodstream of critically ill patients and to identify correlations with clinical and biological parameters. Blood samples were collected from intubated patients at the time of ICU admission from April to June 2011. To identify NETs, DNA and histone H3 were visualized simultaneously by immunofluorescence in blood smears. Cit-H3 was detected using a specific antibody. We assessed relationships of the presence of NETs and Cit-H3 with the existence of bacteria in tracheal aspirate, SIRS, diagnosis, WBC count, and concentrations of IL-8, TNF-α, cf-DNA, lactate, and HMGB1. Forty-nine patients were included. The median of age was 66.0 (IQR: 52.5–76.0) years. The diagnoses included trauma (7, 14.3%), infection (14, 28.6%), resuscitation from cardiopulmonary arrest (8, 16.3%), acute poisoning (4, 8.1%), heart disease (4, 8.1%), brain stroke (8, 16.3%), heat stroke (2, 4.1%), and others (2, 4.1%). We identified NETs in 5 patients and Cit-H3 in 11 patients. NETs and/or Cit-H3 were observed more frequently in “the presence of bacteria in tracheal aspirate” group (11/22, 50.0%) than in “the absence of bacteria in tracheal aspirate” group (4/27, 14.8%) (p<.01). Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3. The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation. NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.

Origin of Circulating Free DNA in Sepsis: Analysis of the CLP Mouse Model

Recently, it has been reported that circulating free DNA (cf-DNA) in the blood is increased in various infectious diseases, including sepsis. Moreover, a relationship between cf-DNA and neutrophil extracellular traps (NETs) has been suggested. However, it is still unclear what the source and physiological role of cf-DNA in sepsis are. In this study, we examined the source of cf-DNA by detecting citrullinated histone H3, a characteristic feature of NET formation, in cecal ligation and puncture- (CLP-)operated mice. In addition, neutrophil depletion using anti-Ly6G antibodies was performed to assess the association between neutrophils and cf-DNA. Increased cf-DNA levels were observed only in CLP mice and not in the control groups; the qPCR findings revealed that the cf-DNA was mainly host-derived, even in bacteremic conditions. Citrullinated histone H3 was not increased in the neutrophils upon CLP, and the depletion of neutrophils showed limited effects on decreasing the amount of cf-DNA. Taken together, these results suggested that elevated cf-DNA levels during early-phase sepsis may represent a candidate biomarker for the severity of sepsis and that, contrary to previous findings, cf-DNA is not derived from neutrophils or NETs.

Case of invasive nontypable Haemophilus influenzae respiratory tract infection with a large quantity of neutrophil extracellular traps in sputum.

Haemophilus influenzae type b was once the most common cause of invasive H. influenzae infection, but the incidence of this disease has decreased markedly with introduction of conjugate vaccines to prevent the disease. In contrast, the incidence of invasive infection caused by nontypable H. influenzae has increased in the US and in European countries. Neutrophil extracellular traps (NETs) are fibrous structures released extracellularly from activated neutrophils during inflammation, including in pneumonia, and rapidly trap and kill pathogens as a first line of immunological defense. However, their function and pathological role have not been fully investigated. Here, we report a case of fatal nontypable H. influenzae infection with severe pneumonia and bacteremia in an adult found to have a vast amount of NETs in his sputum. The patient had a two-day history of common cold-like symptoms and was taken to the emergency room as a cardiopulmonary arrest. He recovered temporarily, but died soon afterwards, although appropriate antibiotic therapy and general management had been instituted. Massive lobular pneumonia and sepsis due to nontypable H. influenzae was found, in spite of H. influenzae type b vaccine being available. His sputum showed numerous bacteria phagocytosed by neutrophils, and immunohistological staining indicated a number of NETs containing DNA, histone H3, and neutrophil elastase. This case highlights an association between formation of NETs and severe respiratory and septic infection. An increase in severe nontypable H. influenzae disease can be expected as a result of “pathogen shift” due to increased use of the H. influenzae type b vaccine in Japan.

Prevalence of, and risk factors for carriage of carbapenem-resistant Enterobacteriaceae among hospitalized patients in Japan

BACKGROUND The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has been reported to be lower in Japan than in many other countries. However, extensive surveillance for CRE carriage has not been performed in Japan. AIM To investigate the prevalence of CRE carriage in Japan among convalescent patients considered to be at high risk of being CRE carriers using an improved selective culture medium. METHODS A cross-sectional survey was conducted in 22 acute care hospitals (ACHs) and 21 long-term care hospitals (LTCHs) in northern Osaka from December 2015 to January 2016. Patients who used incontinence aids, an enteral feeding tube or a urinary catheter were enrolled. Faecal specimens were examined using the newly developed M-ECC for imipenemase (IMP)-producing CRE, which is the most prevalent form of CRE in Japan. The positive isolates were analysed by polymerase chain reaction and sequencing. Risk factors associated with carriage were analysed by logistic regression. FINDINGS Among 1507 patients, 184 (12.2%) carried CRE. The percentage of positive patients was significantly higher in LTCHs (14.9%) than in ACHs (3.6%) (P<0.001). Risk factors for CRE carriage were longer hospital stay [odds ratio (OR) 2.59; 95% confidence interval (CI) 1.87-3.60], enteral feeding (OR 3.03, 95% CI 2.08-4.42) and antibiotic exposure (OR 2.00, 95% CI 1.40-2.87). Among the 233 CRE isolates identified, 223 were IMP producers; the remaining isolates did not produce carbapenemase. CONCLUSIONS This is the first Japanese report to demonstrate the significant spread of CRE in both ACHs and LTCHs using an improved selective medium. A coordinated regional approach may help to prevent further spread.

Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar

The bacterial enzyme New Delhi metallo-β-lactamase hydrolyzes almost all β-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-β-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1), IncX3 plasmids harboring blaNDM-4 (n = 2) or blaNDM-7 (n = 1), IncFII plasmids harboring blaNDM-4 (n = 1) or blaNDM-5 (n = 3), and a multireplicon F plasmid harboring blaNDM-5 (n = 1). Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

Development of selective medium for IMP-type carbapenemase-producing Enterobacteriaceae in stool specimens

BackgroundIdentification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE.MethodsFifteen reference CPE strains producing different types of β-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO4) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO4). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium.ResultsAll tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%).ConclusionsM-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE.

Herpes zoster laryngitis in a patient treated with fingolimod

Development of treatment with immunomodulatory agents has improved prognosis of various autoimmune-related diseases. A sphingosin-1-phosphate receptor modulator, or fingolimod, is the first licensed oral drug for relapsing-remitting multiple sclerosis. The agent reduces circulating lymphocytes by trapping T cells in lymph nodes, possibly leading to reactivation of latent viruses. A 41-year-old Japanese woman who had been treated with fingolimod for 2 years presented with unilateral sore throat. Laryngoscopy revealed exudates unilaterally emerging on the left side of her supraglottic region. Serum level of the varicella zoster virus (VZV)-specific IgG was markedly elevated, and a result of genome sequence using the exudates demonstrated VZV as a possible causative pathogen. Fingolimod therapy was discontinued and the patient was successfully treated with intravenous acyclovir. This is the first reported case of fingolimod-associated herpes zoster laryngitis, in which the local VZV reactivation was demonstrated by next-generation sequencing technology. The present case highlights that the occurrence of VZV reactivation should be recalled in any patients undergoing fingolimod therapy.

PCR-Dipstick Chromatography for Differential Detection of Carbapenemase Genes Directly in Stool Specimens

ABSTRACT A PCR-dipstick chromatography technique was designed and evaluated for differential identification of blaNDM, blaKPC, blaIMP, and blaOXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.

Clinical Specimen-Direct LAMP: A Useful Tool for the Surveillance of blaOXA-23-Positive Carbapenem-Resistant Acinetobacter baumannii

Healthcare-associated infections are a leading cause of morbidity and mortality worldwide. Treatment is increasingly complicated by the escalating incidence of antimicrobial resistance. Among drug-resistant pathogens, carbapenem-resistant Acinetobacter baumannii (CRAb) is of increasing concern because of the limited applicable therapies and its expanding global distribution in developed countries and newly industrialized countries. Therefore, a rapid detection method that can be used even in resource-poor countries is urgently required to control this global public health threat. Conventional techniques, such as bacterial culture and polymerase chain reaction (PCR), are insufficient to combat this threat because they are time-consuming and laborious. In this study, we developed a loop-mediated isothermal amplification (LAMP) method for detecting bla OXA-23-positive CRAb, the most prevalent form of CRAb in Asia, especially in Thailand, and confirmed its efficacy as a surveillance tool in a clinical setting. Clinical samples of sputum and rectal swabs were collected from patients in a hospital in Bangkok and used for LAMP assays. After boiling and centrifugation, the supernatants were used directly in the assay. In parallel, a culture method was used for comparison purposes to evaluate the specificity and sensitivity of LAMP. As a first step, a total of 120 sputum samples were collected. The sensitivity of LAMP was 88.6% (39/44), and its specificity was 92.1% (70/76) using the culture method as the “gold standard”. When surveillance samples including sputum and rectal swabs were analyzed with the LAMP assay, its sensitivity was 100.0%. This method enables the direct analysis of clinical specimens and provides results within 40 minutes of sample collection, making it a useful tool for surveillance even in resource-poor countries.

Pneumorachis associated with multiorgan infection due to Citrobacter koseri

Pneumorachis rarely occurs after spreading from a contiguous site of infection or after a traumatic event. We describe an adult patient who developed sepsis and a renal abscess due to Citrobacter koseri, and computed tomographic imaging identified gas within the entire spinal canal as well as an iliopsoas abscess. This patient recovered from pneumorachis caused by disseminated infection.