Development of rationally designed nucleic acid signatures for microbial pathogens

The detection and identification of microbial pathogens are critical challenges in clinical medicine and public health surveillance. Advances in genome analysis technology are providing an unprecedented amount of information about bacterial and viral organisms, and hold great potential for pathogen detection and identification. In this paper, a rational approach to the development and application of nucleic acid signatures is described based on phylogenetically informative sequence features, especially single nucleotide polymorphisms. The computational tools that are available to enable the development of the next generation of microbial molecular signatures for clinical diagnostics and infectious disease surveillance are reviewed and the impact on public health and national security will be discussed.     

Early events in innate immunity in the recognition of microbial pathogens

Innate immunity is characterised by a rapid action of host effector molecules and leukocytes aimed at limiting the multiplication of invading microbial organisms and destroying them. The recognition and destruction of microorganisms involves humoral factors (e.g., the complement system and natural antibodies) and different cell types (e.g., phagocytic cells, mast cells, natural killer cells). Microbial detection by cells involves germ line-encoded pattern-recognition receptors such as Toll-like receptors and nucleotide-binding oligomerization domain-like receptors. Cellular activation by pathogens leads to the release of antimicrobial peptides (e.g., defensins and peptidoglycan recognition proteins) and cytokines that orchestrate the anti-infectious response. Cytokines enhance phagocytosis and leukocyte microbicidal activity, allow cellular recruitment into the infectious focus, boost hematopoiesis, induce fever and lead to the production of acute phase proteins.     

The use of micromethods for the identification of Vibrios]

The authors recommend micromethods for laboratory studies of Vibrio; such methods may be widely used at bacteriologic laboratories for examinations of biochemical characteristics of these microorganisms, for rapid identification of V. cholerae 01, and for serologic identification (typing) of V. cholerae non 01, since they accelerate the diagnosis and are much simpler than macromethods.     

Early events in innate immunity in the recognition of microbial pathogens

Innate immunity is characterised by a rapid action of host effector molecules and leukocytes aimed at limiting the multiplication of invading microbial organisms and destroying them. The recognition and destruction of microorganisms involves humoral factors (e.g., the complement system and natural antibodies) and different cell types (e.g., phagocytic cells, mast cells, natural killer cells). Microbial detection by cells involves germ line-encoded pattern-recognition receptors such as Toll-like receptors and nucleotide-binding oligomerization domain-like receptors. Cellular activation by pathogens leads to the release of antimicrobial peptides (e.g., defensins and peptidoglycan recognition proteins) and cytokines that orchestrate the anti-infectious response. Cytokines enhance phagocytosis and leukocyte microbicidal activity, allow cellular recruitment into the infectious focus, boost hematopoiesis, induce fever and lead to the production of acute phase proteins.     

Molecular applications for identifying microbial pathogens in the post-9/11 era

Rapid advances in molecular and optical technologies over the past 10 years have dramatically impacted the way biologic research is conducted today. Examples include microarrays, capillary sequencing, optical mapping and real-time sequencing (Pyrosequencing). These technologies are capable of rapidly delivering massive amounts of genetic information and are becoming routine mainstays of many laboratories. Fortunately, advances in scientific computing have provided the enormous computing power necessary to analyze these enormous data sets. The application of molecular technologies should prove useful to the burgeoning field of microbial forensics. In the post-9/11 era, when securing America's food supply is a major endeavor, the need for rapid identification of microbes that accidentally or intentionally find their way into foods is apparent. The principle that distinguishes a microbial forensic investigation from a molecular epidemiology study is that a biocrime has been committed. If proper attribution is to be attained, a link must be made between a particular microbe in the food and the perpetrator who placed it there. Therefore, the techniques used must be able to discriminate individual isolates of a particular microbe. A battery of techniques in development for distinguishing individual isolates of particular foodborne pathogens is discussed.     

Discriminative power of fatty acid methyl ester (FAME) analysis using the microbial identification system (MIS) for Candida (Torulopsis) glabrata and Saccharomyces cerevisiae

Candida (Torulopsis) glabrata is frequently isolated in cases of fungal infection and commonly shows acquired or innate fluconazole resistance. Saccharomyces cerevisiae, an emerging opportunistic yeast pathogen, causes serious systemic infections in immunocompromised, and vaginitis and superficial infections in immunocompetent patients. For both species reliable identification in the routine laboratory is mandatory, but species identification of strains, e.g. trehalose-negative C. glabrata, may be difficult. Therefore, gas-liquid chromatography (GLC) of whole cell fatty acid methyl ester (FAME) profiles, that is independent of assimilation profiles of strains and suitable for reliable and rapid identification of clinically important yeasts, was applied. However, frequent misidentification of C. glabrata as S. cerevisiae has been reported when using the Yeast Clinical Database of MIS. Accuracy of MIS identification may be strongly influenced by the amounts of cell mass analyzed. Therefore, the present study compared the MIS results of these two yeasts achieved with different cell masses. Primarily we optimized, especially with respect to cost-effectiveness, the recommended streaking technique yielding a maximal recovery of 90-130 mg of cell mass from one plate, enabling testing of poor growing strains of C. glabrata. For all C. glabrata strains tested (n = 10) the highest identification scores (SI [Similarity Index] range 0.525-0.963, median 0.832) were achieved with 30 to 45 mg of cell mass. Only 5 of 10 S. cerevisiae strains revealed good library comparisons (SI > or = 0.5) when using 30 mg of cell mass, whereas with 45 mg all strains but two revealed this SI-level. For S. cerevisiae a higher amount of cell mass processed (up to 90 mg) was correlated with better identification scores (SI range using 90 mg: 0.464-0.870, median, 0.737). Several passages prior to FAME analysis of C. glabrata strains on recommended media revealed narrowing of SI ranges, but differences in SI values were not statistically significant.     

Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes

We developed real-time polymerase chain reaction (PCR) assays for rapid detection of the most common and clinically relevant bacteria in positive blood culture bottles, including Staphylococcus spp., S. epidermidis, S. aureus, Enterococcus spp. (including differentiation of E. faecalis and E. faecium), Streptococcus spp., Streptococcus agalactiae, Enterobacteriaceae, E. coli, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp., Bacteroides spp., Haemophilus influenzae, and Neisseria meningitidis. A total of 507 positive blood cultures were investigated according to a specific PCR algorithm based on the microscopic result of the blood culture, and the PCR results were compared to the culture results. Apart from-cross reactions between E. coli and Chryseomonas luteola and Enterococcus faecium and E. durans, the PCR assay correctly identified all bacteria in the blood cultures and did not show any false-positive results. Regarding blood cultures positive with a single species of bacteria (n = 474), 98.3% of all bacteria were correctly detected by the PCR algorithm within a few hours. However, in mixed infections, the sensitivity was lower. The PCR algorithm is well suited for rapid identification of the most common bacteria in positive blood cultures.     

The alcohol use disorders identification test (AUDIT): validation of an instrument for enhancing nursing practice in Hong Kong

This paper describes the psychometric analysis of the alcohol use disorders identification test (AUDIT) after it was modified for use in Hong Kong and administered to examine the patterns of hazardous and harmful drinking. The modified version of AUDIT was an 18-item instrument in which 10 items were completely adopted from the original version and 8 items were added to improve its cultural sensitivity. It was translated into Chinese and back translation was undertaken to confirm the equivalence of the Chinese and English versions. Following a pilot study the instrument was administered to 450 subjects who were recruited from two acute general hospitals, a University Health Clinic and three community health centres. The content validity was judged as adequate by a panel of five international and local experts and the instrument achieved a high reliability coefficient of 0.99 during a test-retest procedure conducted with 20 subjects. Factor analysis was performed on the responses obtained from 450 subjects which supported the construct validity of the 18-item instrument. The modified instrument had a consistently high internal consistency reliability (Cronbach's alpha=0.96-0.97) when tested in the different settings. It was found that a higher percentage of respondents from the hospitals (14.5%) drank at a hazardous or harmful levels compared to those from the community (6.2%) or the University (5.3%). The AUDIT proved a reliable and valid measure with potential applications in Chinese cultures. Early intervention and identification of 'at risk' drinking by the AUDIT is supported as a strategy to be implemented by nurses in primary and secondary health care settings in Hong Kong, where there are indications of increasing alcohol overuse.     

The role of toll-like receptors (TLRs) in pan-cancer

BackgroundToll-like receptors (TLRs) are important components of the innate and adaptive immune systems, and abnormal TLR expression has been linked to a variety of cancers. However, there was a lack of clarity on the association of TLR stimulation with the carcinogenesis of cancer. The study''s goal was to analyse the clinical importance of TLRs expression at the mRNA level in pan-cancer datasets, as well as the link between TLR expression and carcinogenesis, progression, and clinical prognosis.MethodsThe expression profile of TLRs derived from UCSC pan-cancer data was analysed in multiple dimensions, including clinical analysis, immunological subtype analysis, tumour microenvironment (TME) analysis, tumour stem cell correlation analysis, and drug sensitivity analysis. Additionally, we analyse protein-protein interactions, functional enrichment, and chromatin accessibility, as well as TLR expression in single-cell sequencing data.ResultsOur multi-omics analysis results imply that TLRs may operate as a biological marker for carcinogenesis and progression, a potential target for anti-tumour therapy, and a prognostic biomarker, laying the theoretical groundwork for future translational medicine research.ConclusionTLRs are involved in the formation of malignancies and can be explored in further detail as potential prognostic indicators.

Key Messages

• Toll-like receptors (TLRs) are key factors in the process of the innate and adaptive immune response, and their aberrant expression of TLRs have been widely reported in various cancer. However, the association between TLRs stimulation and tumorigenesis of cancer has not been well clarified.
• In this study, in the pan-cancer data, integrated TLR family gene expression analysis, clinical correlation analysis, immune subtype correlation analysis, tumour microenvironment correlation analysis, tumour stem cell correlation analysis, and drug sensitivity correlation analysis were performed.
• TLRs play an important role in the development of tumours and can be studied in depth as potential prognostic markers.

     

病原体重测序芯片用于病原体的检测和公共卫生监测

RPM是一种新的基于微阵列DNA芯片的病原体检测与鉴定技术,可以同时检测一份样品中的多种病原体,对检测到的病原体基因进行测序,并鉴定被检测出的病原体是否与目前已知、未知菌株或变异株具有相似性,从而将医学诊断与公共卫生普查、监控及暴发株的监察联系起来.     

血培养病原菌分布及污染菌判定的实验室检查

目的通过回顾该院的血培养病原菌检出情况以及污染菌的判定分析,了解该院血流感染的主要病原菌构成比,并证实开展双侧双瓶采血检测等实验室检查方法,能有效提高阳性率和血培养污染菌的判定。方法对该院2010年7～12月的1 121份血培养标本进行回顾性分析,了解其病原菌分布,对部分患者开展双侧双瓶采血检测,以血培养中最常见的污染菌凝固酶阴性葡萄球菌(CNS)为例,评价该方法对血培养污染菌判定的价值。结果血培养阳性检出率为10.0%,主要检出菌种为大肠埃希菌,占25.0%,临床分布和时间分布显示为散发,未见院内感染趋势。其中采用双侧双瓶采血检测的血培养标本98例,阳性检出率达22.5%,高于单侧单瓶采血(9.6%),其中检出CNS的比例为22.7%,双瓶48h内同时检出CNS仅为4.5%,而单瓶检出占18.2%,提示临床CNS可能为污染菌,建议结合患者临床资料、状态综合判断,指导用药。结论血培养检出病原菌种类仍以大肠埃希菌为最高,同往年相似。开展采用双侧双瓶采血方法对判定CNS是否为污染菌有一定作用,可提示临床结合患者具体情况综合判断,减少不合理用药。     

Use of carbohydrate and nitrate assimilations in the identification of dematiaceous fungi

A total of 61 isolates of dematiaceous fungi, including Exophiala jeanselmei, Wangiella dermatitidis, Fonsecaea pedrosoi, Phialophora verrucosa, and a few isolates of related organisms were evaluated for their ability to assimilate 13 carbohydrates and sodium nitrate. Results indicated that patterns of assimilations can facilitate specific identifications when used with microscopic morphologic features. Eleven isolates of W. dermatitidis demonstrated negative results for nitrate assimilation, although most of the other fungi tested had positive reactions. The tests did aid in separating this very complex group of fungi.     

Elucidation of the vasoactive intestinal peptide pharmacophore for VPAC(2) receptors in human and rat and comparison to the pharmacophore for VPAC(1) receptors

Vasoactive intestinal peptide (VIP) functions as a neurotransmitter involved in a number of physiological and pathological conditions. The actions of VIP are mediated through VPAC(1) and VPAC(2). In contrast to VPAC(1), which has been extensively studied, little is known about the pharmacology of VPAC(2). In this study we investigated the VIP pharmacophore for VPAC(2) by using alanine and D-amino acid scanning. We found significant species differences, and the human VPAC(2) (hVPAC(2)) expressed in Chinese hamster ovary (CHO) cells, which have been used in previous studies, differed significantly from the native hVPAC(2) in Sup T(1) cells and hVPAC(2) expressed in PANC1 cells. There was a close agreement between binding affinities and potencies for VPAC(2) activation. The amino acids whose backbone or side chain orientations were most important for high affinity potency are Asp(3), Phe(6), Thr(7), Tyr(10), Arg(12), Tyr(22), and Leu(23), whereas the side chains of Ser(2), Asp(8), Asn(9), Gln(16), Val(19), Lys(20), Lys(21), Asn(24), and Ser(25) are not essential. Comparison of the VIP pharmacophore between hVPAC(1) and hVPAC(2) demonstrated that the side chains of Thr(7), Tyr(10), Thr(11), and Tyr(22) were much more critical for high affinity for the hVPAC(2) than the hVPAC(1). In contrast, the orientation of the side chain of Asn(24) was more important for high affinity for the hVPAC(1). This study shows that in assessing the pharmacophore of VIP analogs for the VPAC(2), important species differences need to be considered as well as the expression system used. These results of our study should be useful for designing VPAC subtype-selective analogs, simplified analogs, and possibly metabolically stable analogs.     

全自动细菌鉴定仪直接鉴定血培养阳性标本的临床应用

目的探讨全自动细菌鉴定仪对血培养阳性标本直接鉴定和药敏试验的临床应用价值及其与常规法（转种后取得的纯菌落上机鉴定及药敏试验）的符合率。方法将血培养阳性标本的培养液混匀于专用接种水中,接种于专用鉴定板上直接上机做细菌鉴定和药敏试验,并与转种后取得的纯菌落上机鉴定及药敏结果进行比较。结果86例血培养阳性标本直接上机与转种菌落上机鉴定结果差异无统计学意义（P〈0．01）,血培养阳性标本直接上机与转种菌落上机的药敏试验结果符合率大于95．5％。而直接法比常规法约提前24h。结论对血培养阳性标本进行直接鉴定及药敏试验缩短了检验时间,为菌血症的治疗争取了时间,且与常规法的实验结果符合率高。