呼吸道感染临床实验室检查标本的评价

急性呼吸道感染(RTIs)是临床常见多发病,其病原体对抗生素耐药率的增加对诊断微生物学构成了严峻的挑战。临床微生物室对感染病原体的检出率较低,一般只有50%,且不同部位感染所采集的标本,其结果差异很大。本研究将评述呼吸道感染实验室检查标本采集的影响因素和常见病原体肺炎链球菌、流感嗜血杆菌、肺炎支原体、卡他莫拉菌、肺炎衣原体、嗜肺性军团病杆菌、呼吸道病毒、尿抗原试验的临床意义。     

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal colonization. The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.     

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4⁺ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4⁺ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-α/β cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.     

Fungal pollution of indoor environments and its management

Indoor environments play important roles in human health. The health hazards posed by polluted indoor environments include allergy, infections and toxicity. Life style changes have resulted in a shift from open air environments to air tight, energy efficient, environments, in which people spend a substantial portion of their time. Most indoor air pollution comes from the hazardous non biological agents and biological agents. Fungi are ubiquitous in distribution and are a serious threat to public health in indoor environments. In this communication, we have reviewed the current status on biotic indoor air pollution, role of fungi as biological contaminants and their impact on human health.     

武汉地区医院感染葡萄球菌的耐药性监测

目的了解武汉地区医院感染葡萄球菌的耐药现状。方法采用回顾性分析方法,对2003年1月到2007年12月我院分离的1373株金黄色葡萄球菌和259株表皮葡萄球菌的耐药性进行分析。药敏试验采用K—B纸片法,判断标准根据美国临床实验室标准化委员会（NCCLS）的标准。结果2003年1月到2007年12月我院分离到金黄色葡萄球菌1373株,其中耐甲氧西林的金黄色葡萄球菌（MRSA）有697株,对甲氧西林敏感株（MSSA）有587株,表皮葡萄球菌有259株,其中耐甲氧西林的表皮葡萄球菌（MRSE）有92株,对甲氧西林敏感株（MSSE）有142株。MRSA、MRSE对临床常用的抗生素几乎均耐药,只有对万古霉素和替考拉宁100％敏感;MSSA、MSSE对临床常用抗生素较敏感,但是对青霉素和红霉素耐药率均大于70％。结论武汉地区医院感染MRSA和MRSE对大部分临床常用抗生素均已高度耐药,对万古霉素和替考拉宁依然高度敏感。了解医院感染葡萄球菌的耐药状况,对临床合理选用抗生素十分重要。     

Trypanosoma musculi with Trichinella spiralis or Heligmosomoides polygyrus: concomitant infections in the mouse

Inbred mice infected with Trypanosoma musculi displayed wide variations in peak blood parasitemia. The most susceptible mice were C3H and A strain, while Balb/c, C57B1/6, and the related congenic B10 strains were the most resistant. The effect of an intestinal infection with either Trichinella spiralis or Heligmosomoides polygyrus on proliferation of T. musculi was investigated. T. spiralis infections given at the same time or up to 45 days before a T. musculi infection always caused an increase in blood parasitemia in C3H mice. Maximum increases were observed when T. spiralis infections preceded T. musculi by 5-10 days. In all mouse strains examined, dual infections increased maximum parasitemia by two- to four-fold, regardless of the degree of resistance of that mouse strain to either T. musculi or T. spiralis. This suggested that the immunological "cost" of a T. spiralis infection was the same for strains that were strong or weak responders to a primary infection with T. spiralis. In contrast, infection with H. polygyrus did not promote T. musculi parasitemia over the level of a single infection. The increase in blood parasitemia in T. spiralis-infected mice was largely due to the intestinal adult worm, but migratory larvae and mature muscle larvae also stimulated increased parasitemias. The increase in parasitemia was proportionate to the dose of T. spiralis, and the sex of the host did not affect the blood trypanosome level.     

Trypanosoma musculi and Trichinella spirails: Concomitant infections and selection for resistance genotypes in mice

Trypanosoma musculi infections were given to mice of different strains before, at the same time, and after an infection with 400 Trichinella spiralis. Examined parameters of the host response to T. spiralis were worm rejection, antifecundity responses, development of immunological memory, and muscle larvae burden. After dual infection, each mouse strain showed characteristic effects on resistance to T. spiralis. This was due to a dynamic interaction between the genes controlling rejection of T. spiralis and those influencing T. musculi growth. C3H mice develop high trypanosome parasitemias. This impairs worm expulsion and the development of memory to T. spiralis when Trypanosoma infections take place on the same day or 7 days before. The C57B1/6 mouse develops low parasitemias and T. musculi infections on the same day, or 7 days before T. spiralis, delaying worm rejection only slightly despite the overall weak capacity of B6 mice to expel worms. NFR-strain mice are strong responders to T. spiralis and also develop low parasitemias. Trypanosome infections on the same day, or after T. spiralis, produce a delay in worm rejection; the former is comparable to C3H mice. However, NFR mice alone showed enhanced rejection of worm when T. musculi infections preceded T. spiralis by 7 days. An unusual feature of C3H mice was that T. musculi infections 7 days before T. spiralis increased antifecundity responses at the same time that worm expulsion was inhibited. Trypanosome infections can therefore modulate distinct antihelminth immune responses in different directions simultaneously. The different outcomes of dual infections compared with single infections provides another selective mechanism by which genetic polymorphisms can be established and maintained in the vertebrate host.     

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions¹. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.     

Control of transmission with two types of infection

The effectiveness of a vaccination strategy to control transmission of an infectious disease depends on the way vaccine doses are distributed to individuals in a community of households. Here we show that this dependence is more complicated when acquisition and severity of illness are determined by the size of the infecting dose, as is thought to be the case for measles and varicella. Two alternative formulations for the way vaccination changes an individual's susceptibility and infectivity show that vaccination coverage, the nature of the vaccine response and the distribution of household size also have a big impact on which strategy is more effective. These judgements are made by comparing the post-vaccination reproduction numbers corresponding to different vaccination strategies.     

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4'',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.