Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.     

新生儿感染性肺炎的病原茵状况分析

目的了解本地区新生儿感染性肺炎的病原菌的菌种、构成比及耐药情况,探索临床合理选用抗生素。方法细菌鉴定及药敏试验采用VITEK-60全自动细菌鉴定仪。结果本地区新生儿感染性肺炎的病原菌主要为革兰阴性杆菌（92．81％）,其中以肺炎克雷伯菌最为常见,革兰阳性球菌感染较少（7．19％）。革兰阴性杆菌对头孢二代、三代和氨基糖苷类抗生素的耐药率均较高,对喹诺酮类抗生素耐药率较低。亚胺培南具有良好的抗菌活性。结论肺炎克雷伯菌是本地区新生儿感染性肺炎的主要病原菌。经验性治疗用药可首选亚胺培南、头孢替坦、环丙沙星等,建议临床根据药敏结果选用抗生素。     

Subdivision meshes for organizing spatial biomedical data

As biomedical images and volumes are being collected at an increasing speed, there is a growing demand for efficient means to organize spatial information for comparative analysis. In many scenarios, such as determining gene expression patterns by in situ hybridization, the images are collected from multiple subjects over a common anatomical region, such as the brain. A fundamental challenge in comparing spatial data from different images is how to account for the shape variations among subjects, which make direct image-to-image comparisons meaningless. In this paper, we describe subdivision meshes as a geometric means to efficiently organize 2D images and 3D volumes collected from different subjects for comparison. The key advantages of a subdivision mesh for this purpose are its light-weight geometric structure and its explicit modeling of anatomical boundaries, which enable efficient and accurate registration. The multi-resolution structure of a subdivision mesh also allows development of fast comparison algorithms among registered images and volumes.     

Towards radiological diagnosis of abdominal adhesions based on motion signatures derived from sequences of cine-MRI images

This paper reports novel development and preliminary application of an image registration technique for diagnosis of abdominal adhesions imaged with cine-MRI (cMRI). Adhesions can severely compromise the movement and physiological function of the abdominal contents, and their presence is difficult to detect. The image registration approach presented here is designed to expose anomalies in movement of the abdominal organs, providing a movement signature that is indicative of underlying structural abnormalities. Validation of the technique was performed using structurally based in vitro and in silico models, supported with Receiver Operating Characteristic (ROC) methods. For the more challenging cases presented to the small cohort of 4 observers, the AUC (area under curve) improved from a mean value of 0.67 ± 0.02 (without image registration assistance) to a value of 0.87 ± 0.02 when image registration support was included. Also, in these cases, a reduction in time to diagnosis was observed, decreasing by between 20% and 50%. These results provided sufficient confidence to apply the image registration diagnostic protocol to sample magnetic resonance imaging data from healthy volunteers as well as a patient suffering from encapsulating peritoneal sclerosis (an extreme form of adhesions) where immobilization of the gut by cocooning of the small bowel is observed. The results as a whole support the hypothesis that movement analysis using image registration offers a possible method for detecting underlying structural anomalies and encourages further investigation.     

血清胱抑素C 水平变化在支原体肺炎早期肾损伤的临床预防价值

目的:探讨血清胱抑素C水平变化对小儿支原体肺炎早期肾损伤的临床预防价值,并依据胱抑素C水平变化制定支原体肺炎患儿的临床治疗疗程及康复时间,以减少支原体肺炎的复发。方法:对临床确诊的129例为支原体肺炎的住院患儿进行胱抑素C、尿素氮、肌酐的检测。结果:肺炎支原体患儿胱抑素C水平较对照组明显升高,临床病愈患儿中有39例胱抑素C水平较正常对照组高,并有统计学意义,尿素氮水平及肌酐与正常对照组比较无明显差异,39例胱抑素C水平高的患儿在随访中有27例再次感染支原体肺炎,而90例随访结果正常者仅有9例复发支原体肺炎入院。结论:对肺炎支原体患儿进行胱抑素C的检测有助于预知肺炎支原体对肾脏的早期肾损伤及对支原体肺炎患儿的愈后判断并进行早期干预预防再次感染复发。     

Incidence of leukaemia in young people in the vicinity of Hinkley Point nuclear power station, 1959-86.

The incidence of leukaemia and non-Hodgkin''s lymphoma in young people (aged under 25) living in a predefined area around the nuclear power station at Hinkley Point, Somerset, was examined for the period 1959-86 by using cancer registry data. During the period since Hinley Point began operations--that is, 1964-86--there were 19 cases in the area compared with 10.4 expected from national rates, giving a standardised registration ratio of 1.82 (95% confidence interval 1.10 to 2.85). The incidence in the rest of Somerset was also high, however (standardised registration ratio 1.18; 95% confidence interval 0.98 to 1.41), and the high rate around Hinkley Point may simply have been reflecting the high local incidence (ratio of the two standardised registration ratio''s 1.54; 95% confidence interval 0.90 to 2.52). Analysis of predetermined five year periods showed that the excess cases in the Hinkley Point area were concentrated in the 10 years 1964-73 after commissioning of the station, at a time when rates in the rest of Somerset were close to the national average. In particular the nine cases occurring in the five years 1969-73 were about four times the number expected from national rates (standardised registration ratio 3.96; 95% confidence interval 1.81 to 7.52). Rates in the Hinkley Point area after 1973 were fairly low, especially as compared with the rest of Somerset. In the five years 1959-63 (that is, before Hinkley Point was commissioned) rates throughout Somerset (including the Hinkley Point area) were higher than the national rate. These findings should be interpreted with caution, and further studies are required to test the plausibility of theories relating to radiation and viruses.     

Particulate matter exposure predicts residence in high-risk areas for community acquired pneumonia among hospitalized children

Particulate matter exposure is a risk factor for lower respiratory tract infection in children. Here, we investigated the geospatial patterns of community-acquired pneumonia and the impact of PM_2.5 (particulate matter with an aerodynamic diameter ≤2.5 µm) on geospatial variability of pneumonia in children. We performed a retrospective analysis of prospectively collected population-based surveillance study data of community-acquired pneumonia hospitalizations among children <18 years residing in the Memphis metropolitan area, who were enrolled in the Centers for Disease Control and Prevention sponsored Etiology of Pneumonia in the Community (EPIC) study from January 2010 to June 2012. The outcome measure, residence in high- and low-risk areas for community-acquired pneumonia, was determined by calculating pneumonia incidence rates and performing cluster analysis to identify areas with higher/lower than expected rates of community-acquired pneumonia for the population at risk. High PM_2.5 was defined as exposure to PM_2.5 concentrations greater than the mean value (>10.75 μg/m³), and low PM_2.5 is defined as exposure to PM_2.5 concentrations less than or equal to the mean value (≤10.75 μg/m³). We also assessed the effects of age, sex, race/ethnicity, history of wheezing, insurance type, tobacco smoke exposure, bacterial etiology, and viral etiology of infection. Of 810 (96.1%) subjects with radiographic community-acquired pneumonia, who resided in the Memphis metropolitan area and had addresses which were successfully geocoded (Supplementary Figure F2), 220 (27.2%) patients were identified to be from high- (n = 126) or low-risk (n = 94) community-acquired pneumonia areas. Community-acquired pneumonia in Memphis metropolitan area had a non-homogenous geospatial pattern. PM_2.5 was associated with residence in high-risk areas for community-acquired pneumonia. In addition, children with private insurance and bacterial, as opposed to viral, etiology of infection had a decreased risk of residence in a high-risk area for community-acquired pneumonia. The results from this paper suggest that environmental exposures as well as social risk factors are associated with childhood pneumonia.     

Comparison of techniques for sex determination of American martens

Accurate determination of sex in harvested species is critical for understanding demography and developing population models for management. We used genetic-based sex identification to assess accuracy of external carcass and pelt examination at registration and maximum canine root area (MRA) to determine sex of American martens (Martes americana) trapped in the Upper Peninsula of Michigan, 2000–2004. Overall percent similarity between MRA and genetic-based sex determination was 98.4% (n = 188). In contrast, only 84.6% (n = 421) of martens were similarly classified using external examination. For external examination, percent similarity to genetic-based sex determination for juveniles (<1-yr old) and adults (≥1-yr old; Wald χ²₁ = 2.168, P = 0.141), as well as for males and females (Wald χ²₁ = 0.005, P = 0.946), was similar. We recommend MRA as a suitable technique for sex determination of martens; thus, marten sex and age (using cementum annuli counts) can be obtained from one lower canine tooth. We do not recommend use of external examination at registration to identify sex of martens without implementing additional quality assurance measures. © 2010 The Wildlife Society.     

Experience with mutagenicity testing of new drugs: viewpoint of a regulatory agency

Quality and quantity of mutagenicity testing were analyzed for drugs with new active compounds which were submitted for registration in the Federal Republic of Germany from mid 1982 to mid 1986. A large variety of deficiencies was found, applying to selection and number of mutagenicity tests as well as to test performances. Only 65 out of the 144 drugs submitted for registration were tested sufficiently in the initial phase of registration. From 1982 to 1986 this situation has not been changed markedly. Inadequate test performance still remains the main reason for insufficient testing, leading in some cases to artificially positive results. For in vivo tests the selection of test species was mainly motivated by technical reasons and not by characteristics of the test compound. Most of the insufficiencies were eliminated during the second phase of registration. In some cases insufficient mutagenicity testing led to consequences concerning risk-benefit assessment of the drug and its regulation.     

利奈唑胺治疗儿童社区获得性金黄色葡萄球菌肺炎的临床疗效分析

目的:分析儿童社区获得性金葡菌肺炎应用利奈唑胺的临床疗效.方法:选择2008-1-1至2009-3-1在青岛大学医学院附属医院普通儿科及青岛市儿童医院诊断社区获得性金葡菌肺炎患儿22人为研究对象,分析其体温、咳嗽、肺部罗音、住院时间等临床资料.结果:患儿的平均住院时间为13.0±2.26天,有效率100%,不良反应发生率4.55%.结论:利奈唑胺治疗儿童社区获得性金葡菌肺炎有较高的临床治愈率,可快速改善症状.缩短住院时间.     

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting

Abstract Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time-consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1–16 as well as 21 non- pneumophila isolates could be identified and differentiated at the species level using this system.     

Pathologic anatomy of pneumonia caused by Legionella: on the materials of outbreak in town Verkhnyaya Pyshma

Data on postmortem examination of five patients deceased during Legionnaires' outbreak in town Verkhnyaya Pyshma are presented in the article. Feature of course of the disease was severe affection of the lungs with development of toxic shock. Pathomorphological picture in lungs was characterized by polymorphism, presence of shock reaction and ventilator-associated pneumonia (atelectases, distelectases, tracheobronchitis). In autopsy material from four fatal cases Legionella pneumophila serogroup 1 was detected. Causative agent of pneumonia was not determined in one fatal case although comparison of clinical and morphological characteristics of this case with other four cases as well as detection of Gram-negative bacillus in alveolar macrophages allowed to consider this case of pneumonia as caused by Legionella. In studied fatal cases postmortem diagnoses were ascertained in which main disease was bilateral pneumonia caused by Legionella pneumophila serogroup 1 complicated by toxic shock.     

从临床肺炎死亡树购中分离到致病性大肠埃希菌

目的对6例1月内因肺炎死亡的树鼢采样进行病原菌分离培养鉴定分析。方法解剖死亡树购,利用无菌刀片切开肺组织,用无菌接种环插入肺内采样接种于营养琼脂培养基,另取两份样品进行细菌涂片革兰氏染色和抗酸染色。培养出来的细菌进行进一步分离和菌落生长情况的观察,并经革兰氏染色、抗酸染色、氧化酶试验、生化编码鉴定和9种药敏试验,初步确定树鼢肺部感染的致病菌及其药敏情况。结果样本革兰氏染色见到大量阴性杆菌,抗酸染色结果显示为非结核分枝杆菌,大小约为0．2μm×2～6μm。营养琼脂培养6例样品中均仅见1株旺盛生长的细菌,进一步分离培养经革兰氏染色为阴性杆菌,抗酸染色为非结核分枝杆菌,大小和染色结果与样本涂片相同,经鉴定为致病性大肠埃希菌。药敏试验表明该菌对头孢哌酮,呋喃妥因,氨苄西林,阿米卡星,氧氟沙星,诺氟沙星,磺胺甲嗯唑／甲氧苄定高度敏感;对庆大霉素和青霉素G为低度敏感。结论6例树鼢死亡原因均为细菌性肺炎,病原菌初步鉴定为致病性大肠埃希菌。药敏实验筛选出的药物可为临床治疗树鼢该类病例用药提供指导。     

Detection of Plant DNA in the Bronchoalveolar Lavage of Patients with Ventilator-Associated Pneumonia

Background

Hospital-acquired infections such as nosocomial pneumonia are a serious cause of mortality for hospitalized patients, especially for those admitted to intensive care units (ICUs). Despite the number of the studies reported to date, the causative agents of pneumonia are not completely known. Herein, we found by molecular technique that vegetable and tobacco DNA may be detected in the bronchoalveolar lavage from patients with ventilator-associated pneumonia (VAP).

Methodology/Principal Findings

In the present study, we studied bronchoalveolar lavage (BAL) from patients admitted to ICUs with ventilator-associated pneumonia. BAL fluids were assessed with molecular tests, culture and blood culture. We successfully identified plant DNA in six patients out of 106 (6%) with ventilator-associated pneumonia. Inhalation was confirmed in four cases and suspected in the other two cases. Inhalation was significantly frequent in patients with plant DNA (four out of six patients) than those without plant DNA (three out of 100 patients) (P<0.001). Nicotiana tabacum chloroplast DNA was identified in three patients who were smokers (cases 2, 3 and 6). Cucurbita pepo, Morus bombycis and Triticum aestivum DNA were identified in cases 1, 4 and 5 respectively. Twenty-three different bacterial species, two viruses and five fungal species were identified from among these six patients by using molecular and culture techniques. Several of the pathogenic microorganisms identified are reported to be food-borne or tobacco plant-associated pathogens.

Conclusions/Significance

Our study shows that plants DNA may be identified in the BAL fluid of pneumonia patients, especially when exploring aspiration pneumonia, but the significance of the presence of plant DNA and its role in the pathogenesis of pneumonia is unknown and remains to be investigated. However, the identification of these plants may be a potential marker of aspiration in patients with pneumonia.     

小儿肺炎支原体的分离培养及其药敏试验分析

对356例小儿肺炎患者的咽拭子进行肺炎支原体(MP)分离培养并对9种抗生素进行药敏试验.结果显示,MP阳性55例,阳性率为15.4%,耐药性由高到低为罗红霉素(54.5%)>克拉霉素(32.7%)=阿奇霉素(32.7%)>红霉素(21.8%)=加替沙星(21.8%)>克林霉素(20.0%)>乙酰螺旋霉素(9.1%)>司...     

Diagnostic value of different laboratory methods in the diagnosis of pneumonia caused by Haemophilus influenzae type B

Comparative analysis of the diagnostic value of different laboratory methods in the diagnosis of H. influenzae b (Hib) pneumonia in children (bacteriological method, latex agglutination, counter immunoelectrophoresis, the passive hemagglutination test and the enzyme immunoassay (EIA) was carried out. EIA proved to be the most informative method for the diagnosing Hib pneumonia. EIA makes it possible to detect specific Hib antigens in different clinical materials in 48.8% of cases, as well as high titers of antibodies to mis infective agent in 61.7% of cases. The authors propose the unified criteria of the laboratory diagnosis of Hib infection in children.     

Biosecurity measures in 48 isolation facilities managing highly infectious diseases

Biosecurity measures are traditionally applied to laboratories, but they may also be usefully applied in highly specialized clinical settings, such as the isolation facilities for the management of patients with highly infectious diseases (eg, viral hemorrhagic fevers, SARS, smallpox, potentially severe pandemic flu, and MDR- and XDR-tuberculosis). In 2009 the European Network for Highly Infectious Diseases conducted a survey in 48 isolation facilities in 16 European countries to determine biosecurity measures for access control to the facility. Security personnel are present in 39 facilities (81%). In 35 facilities (73%), entrance to the isolation area is restricted; control methods include electronic keys, a PIN system, closed-circuit TV, and guards at the doors. In 25 facilities (52%), identification and registration of all staff entering and exiting the isolation area are required. Access control is used in most surveyed centers, but specific lacks exist in some facilities. Further data are needed to assess other biosecurity aspects, such as the security measures during the transportation of potentially contaminated materials and measures to address the risk of an "insider attack."     

Rates and risk factors associated with hospitalization for pneumonia with ICU admission among adults

Background

Pneumonia poses a significant burden to the U.S. health-care system. However, there are few data focusing on severe pneumonia, particularly cases of pneumonia associated with specialized care in intensive care units (ICU).

Methods

We used administrative and electronic medical record data from six integrated health care systems to estimate rates of pneumonia hospitalizations with ICU admissions among adults during 2006 through 2010. Pneumonia hospitalization was defined as either a primary discharge diagnosis of pneumonia or a primary discharge diagnosis of sepsis or respiratory failure with a secondary diagnosis of pneumonia in administrative data. ICU admissions were collected from internal electronic medical records from each system. Comorbidities were identified by ICD-9-CM codes coded during the current pneumonia hospitalization, as well as during medical visits that occurred during the year prior to the date of admission.

Results

We identified 119,537 adult hospitalizations meeting our definition for pneumonia. Approximately 19% of adult pneumonia hospitalizations had an ICU admission. The rate of pneumonia hospitalizations requiring ICU admission during the study period was 76 per 100,000 population/year; rates increased for each age-group with the highest rates among adults aged ≥85 years. Having a co-morbidity approximately doubled the risk of ICU admission in all age-groups.

Conclusions

Our study indicates a significant burden of pneumonia hospitalizations with an ICU admission among adults in our cohort during 2006 through 2010, especially older age-groups and persons with underlying medical conditions. These findings reinforce current strategies aimed to prevent pneumonia among adults.

     

Proteomic analysis of exhaled breath condensate for diagnostics of respiratory system diseases

Study of proteomic composition of exhaled breath condensate (EBC) is a promising non-invasive method for diagnostics of respiratory system diseases in patients. In this study the EBC proteomic composition of 53 donors, including patients with different respiratory system diseases has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of proteins have been recognized: proteins found in all pathologies (dermcidin, alpha-1- microglobulin, SHROOM3), proteins simultaneously found in two groups (CSTA, LCN1, JUP, PIP, TXN), and proteins specific for a particular group (PRDX1, annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used for identification of potential protein markers specific for inflammatory pulmonary diseases of infective origin (pneumonia) as well as for non-infectious diseases such as chronic obstructive pulmonary disease (COPD).